A contemporary reassessment of the enhanced transient expression system based on the tombusviral silencing suppressor protein P19

Transient transgenic expression accelerates pharming and facilitates protein studies in plants. One embodiment of the approach involves leaf infiltration of Agrobacterium strains whose T‐DNA is engineered with the gene(s) of interest. However, gene expression during ‘agro‐infiltration’ is intrinsically and universally impeded by the onset of post‐transcriptional gene silencing (PTGS). Nearly 20 years ago, a simple method was developed, whereby co‐expression of the tombusvirus‐encoded P19 protein suppresses PTGS and thus enhances transient gene expression. Yet, how PTGS is activated and suppressed by P19 during the process has remained unclear to date. Here, we address these intertwined questions in a manner also rationalizing how vastly increased protein yields are achieved using a minimal viral replicon as a transient gene expression vector. We also explore, in side‐by‐side analyses, why some proteins do not accumulate to the expected high levels in the assay, despite vastly increased mRNA levels. We validate that enhanced co‐expression of multiple constructs is achieved within the same transformed cells, and illustrate how the P19 system allows rapid protein purification for optimized downstream in vitro applications. Finally, we assess the suitability of the P19 system for subcellular localization studies – an originally unanticipated, yet increasingly popular application – and uncover shortcomings of this specific implement. In revisiting the P19 system using contemporary knowledge, this study sheds light onto its hitherto poorly understood mechanisms while further illustrating its versatility but also some of its limits.