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

Planta
Christopher I Cazzonelli, Jeff Velten

Abstract

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 ...Continue Reading

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Citations

Jul 27, 2007·Transgenic Research·Christopher Ian Cazzonelli, Jeff Velten
Feb 21, 2007·Plant Cell Reports·Sergio Rosales-MendozaAngel Gabriel Alpuche-Solís
Nov 19, 2011·BMC Biotechnology·Wusheng LiuC Neal Stewart
Jul 27, 2010·Journal of Plant Physiology·Sergio Rosales-MendozaSchuyler S Korban
Mar 26, 2016·Frontiers in Bioengineering and Biotechnology·Naomichi FujiuchiRyo Matsuda
Dec 3, 2009·Molecular Plant·Christopher I CazzonelliBarry J Pogson
Mar 9, 2017·Frontiers in Plant Science·Kamila BłażejewskaIzabela A Chincinska

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