Submitted to: Planta
Publication Type: Peer reviewed journal
Publication Acceptance Date: 2/7/2006
Publication Date: 8/1/2006
Citation: Cazzonelli, C.I., Velten, J.P. 2006. An in vivo luciferase-based transient assay system using agrobacteria-infiltration: Implications for post-transcriptional gene silencing. Planta. 224(3):582-597. Interpretive Summary: A new luciferase-based, in vivo transient assay was developed and tested by quantifying plant promoter activities and an examination of factors impacting post transcriptional gene silencing in model plant species.
Technical Abstract: An in vivo assay system for analyzing transient luciferase expression in Agrobactera-infused tobacco leaves is described. The system makes use of Agrobacterium tumefaciens harboring T-DNA vectors containing either an intron-containing firefly (Photinus pyralis) luciferase (EC 188.8.131.52) gene or an intron-containing sea pansy (Renilla reniformis) luciferase (EC 184.108.40.206) gene. Single or mixed Agrobacteria lines are infiltrated into leaf tissues (Nicotiana tabacum or Nicotiana benthamiana) through stomatal openings and leaf discs from infused areas floated on reaction buffers specific to each enzyme. Photons emitted are then measured continuously to determine reporter gene activity. Parameters including: buffer composition; Agrobacteria density; infusion location; reaction timing; and environmental factors (light and temperature) were examined to optimize assay reliability and sensitivity. The resulting in vivo assay system generates results comparable to those obtained using a commercially available in vitro dual-luciferase® 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 (EC 220.127.116.11), p19 and AC2, confirm the occurrence of PTGS within infused zones, and provide a convenient mechanism for PTGS analysis. The in vivo transient assay was used to examine the affect 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 post-transcriptional gene silencing, and the ability of HcPro to suppress PTGS.