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

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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® 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.

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Abbreviations

AC2:

Geminivirus trans-activating protein

alcA :

Aspergillus nidulans alcA (alcohol dehydrogenase) promoter

alcR :

Aspergillus nidulans alcR (activator) gene

bar :

Phosphinothricin acetyl transferase gene

CAM:

Coelenterazine assay mixture

CbCi:

Castor bean catalase intron

dpi:

Days post-infusion

hpi:

Hours post-infusion

HcPro:

Helper component protein

GFP:

Green fluorescent protein (Aequorea victoria)

GUS:

Escherichia coli β-glucuronidase

GUSi:

Intron-modified β-glucuronidase

IM:

Infiltration media

LAM:

Luciferin (firefly) assay mixture

FLUC:

Firefly (Photinus pyralis) luciferase

FiLUC:

Firefly (Photinus pyralis) intron-modified luciferase

p19:

19 kDa protein of tombusviruses

PTGS:

Post-transcriptional gene silencing

PVY:

Potato virus Y

PHV:

Pepper Huasteco virus

R0 :

Primary transformant

R1 :

Progeny of self-fertilized R0 plant

RLUC:

Renilla reniformis (sea pansy) luciferase

RiLUC:

Renilla reniformis (sea pansy) intron-modified luciferase

RLU:

Relative light units

TrAP:

Transcription activator (or transactivator) protein

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Acknowledgements

We would like to thank Dr. S. Luke Mankin (BASF Plant Science, Res Triangle Park, NC, USA) for supplying FiLUC, Dr. Stan Gelvin (Purdue University, West Lafayette, IN, USA) for the pE1778-SUPER promoter vector, and Dr. David Baulcombe (The Sainsbury Laboratory, Norwich, UK) for pBIN61-HcPro and pBIN61-p19 suppressor constructs. We wish to also thank Dr. Robert Gilbertson (University of California, Davis, CA, USA) for pBluescript::PHV-AC2, Syngenta Limited for providing the ethanol-inducible (alcA & alcR) constructs and Dr. William Gurley (University of Florida, Gainesville, FL, USA) for the Gmhsp17.5 heat shock promoter. Our appreciation goes out to John Burke and Junping Chen (USDA-ARS) for critical reading of the manuscript and the pTHSP17.5-E construct, David Wheeler, Brian Sanderson, Kay McCrary and DeeDee Laumbach (USDA-ARS) for expert technical support, and the two anonymous Planta reviewers for suggestions that resulted in a much improved manuscript. Funding was provided by a USDA-ARS post-doctoral fellowship (CIC).

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  1. USDA-ARS (United States Department of Agriculture-Agricultural Research Services), 3810 4th Street, Lubbock, TX, 79415, USA

    Christopher Ian Cazzonelli & Jeff Velten

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  1. Christopher Ian Cazzonelli
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Correspondence to Jeff Velten.

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Cazzonelli, C.I., Velten, J. An in vivo, luciferase-based, Agrobacterium-infiltration assay system: implications for post-transcriptional gene silencing. Planta 224, 582–597 (2006). https://doi.org/10.1007/s00425-006-0250-z

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