Expanding agroinfiltration host range with broad-spectrum effectors monitored by autoluminescent reporter

Abstract Agrobacterium-mediated transient transformation is a fundamental tool for plant research and molecular pharming, but its application is often limited by host immune responses, post-transcriptional gene silencing (PTGS), and the lack of a robust, quantitative reporter for species beyond Nicotiana benthamiana. To address these challenges, we developed a sensitive reporter system based on the fungal bioluminescence pathway (FBP). As an autonomous, substrate-free metabolic pathway, the FBP enables high-throughput, quantitative screening without external manipulations. Using this system, we systematically evaluated factors affecting transformation efficiency across more than 20 plant families. This cross-lineage analysis identified that co-expressing NahG and P19, termed the NaP19 module, not only mitigates P19-induced cytotoxicity but also synergistically enhances transient expression in diverse species, including agroinfiltration-recalcitrant crops and horticultural plants. We demonstrate the platform’s in vivo utility in its native genetic background for applications such as protein localization, interaction assays, and transcriptional regulation studies. By integrating a quantitative, substrate-free reporter with a broadly effective NaP19 enhancer, this work establishes a robust and versatile platform for advancing functional genomics and biotechnology throughout the land plant lineages.