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

SUMMARY: 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. Significance Statement: Since its disclosure in 2003, agro‐infiltration used in combination with the P19 RNA silencing suppressor has been a popular method to achieve high levels of transient gene expression in Nicotiana benthamiana, though the underlying molecular mechanisms have remained unclear. By shedding light on these mechanisms, the present contemporary reassessment of the method allows improvements to protein yields and further illustrates its versatility, but also exposes and rationalizes some of its limits. [ABSTRACT FROM AUTHOR]