An in vivo, luciferase-based, Agrobacterium-infiltration assay system:: implications for post-transcriptional gene silencing

An in vivo assay system for analyzing transient luciferase expression in tobacco leaves infused with Agrobacterium tumefaciens is described. The system makes use of A. tumefaciens harboring T-DNA vectors containing either an intron-containing firefly (Photinus pyralis) luciferase (EC 1.13.12.7) gene or an intron-containing sea pansy (Renilla reniformis) luciferase (EC 1.13.12.5) gene. Single or mixed Agrobacterium lines were infiltrated into leaf tissues (Nicotiana tabacum or Nicotiana benthamiana) through stomatal openings and leaf disks from infused areas floated on reaction buffers specific to each enzyme. Photons emitted were then measured to determine reporter gene activity. Parameters affecting assay reliability and sensitivity were tested, including: buffer composition; bacterial density; infusion location; reaction kinetics; and environmental factors (light and temperature). The resulting in vivo assay system generates results comparable to those obtained using a commercially available in vitro dual-luciferase (R) reporter gene assay, and reports relative expression levels, as well as induction characteristics, analogous to those obtained using leaf tissue from stably transformed plants harboring the same promoter::gene constructs. Light and temperature were observed to markedly impact transient reporter activities. Co-expression of viral suppressors of post-transcriptional gene silencing (PTGS), HcPro, p19 and AC2, confirms the occurrence of PTGS within infused zones, and provides a convenient mechanism for PTGS analysis. The in vivo transient assay was used to examine the effect on PTGS of factors such as: promoter strength; incubation temperature and double-stranded RNA production. Results from these assays provide insight into the mechanism(s) used by plants to trigger and maintain PTGS.