title: Rapid fluorescent reporter quantification by leaf disc analysis and its application in plant-virus studies.
abstract: BACKGROUND
  Fluorescent proteins are extraordinary tools for biology studies due to their versatility; they are used extensively to improve comprehension of plant-microbe interactions. The viral infection process can easily be tracked and imaged in a plant with fluorescent protein-tagged viruses. In plants, fluorescent protein genes are among the most commonly used reporters in transient RNA silencing and heterologous protein expression assays. Fluorescence intensity is used to quantify fluorescent protein accumulation by image analysis or spectroscopy of protein extracts; however, these methods might not be suitable for medium- to large-scale comparisons.
  
  
  RESULTS
  We report that laser scanners, used routinely in proteomic studies, are suitable for quantitative imaging of plant leaves that express different fluorescent protein pairs. We developed a microtiter plate fluorescence spectroscopy method for direct quantitative comparison of fluorescent protein accumulation in intact leaf discs. We used this technique to measure a fluorescent reporter in a transient RNA silencing suppression assay, and also to monitor early amplification dynamics of a fluorescent protein-labeled potyvirus.
  
  
  CONCLUSIONS
  Laser scanners allow dual-color fluorescence imaging of leaf samples, which might not be acquired in standard stereomicroscope devices. Fluorescence microtiter plate analysis of intact leaf discs can be used for rapid, accurate quantitative comparison of fluorescent protein accumulation.
authors: Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, Madrid 28049, Spain.
authors: Fabio
authors: F
authors: Pasin
authors: Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, Madrid 28049, Spain.
authors: Satish
authors: S
authors: Kulasekaran
authors: Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, Madrid 28049, Spain.
authors: Paolo
authors: P
authors: Natale
authors: Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, Madrid 28049, Spain.
authors: Carmen
authors: C
authors: Simón-Mateo
authors: Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, Madrid 28049, Spain.
authors: Juan Antonio
authors: JA
authors: García
keywords: Fluorescence spectroscopy; Fluorescent protein; Microtiter plate; Plant virus; RNA silencing