Rapid fluorescent reporter quantification by leaf disc analysis and its application in plant-virus studies

Abstract

BackgroundFluorescent 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.ResultsWe 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.ConclusionsLaser 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.

CRISPR-TSKO: A Technique for Efficient Mutagenesis in Specific Cell Types, Tissues, or Organs in Arabidopsis

“…The coding sequence for mTagBFP2, based on a previously reported mTagBFP2 (Pasin et al, 2014), was PCR amplified with primers RB42 and RB43 from a synthesized fragment (Gen9) and inserted into a BsaIdigested pGGC000 plasmid via ligation.…”

An atypical RNA silencing suppression strategy provides a snapshot of the evolution of sweet potato-infecting potyviruses

“…For the transient expression experiments, GFP fluorescence intensity quantification was carried out placing individual 5.0 mm-diameter leaf discs in a black 96-well plate (Nunc) filled with 50 µl water/well and acquiring the signal in a monochromator-based plate reader (Infinite M200, Tecan Group) 71 . Four discs per plant were collected.…”

Reverse Genetics and High Throughput Sequencing Methodologies for Plant Functional Genomics

“…Agroinoculation became a reliable method for plant functional gene analysis after being firstly developed as a simple procedure for plant systemic infection with viruses by delivery of viral genomes into plant tissues using Agrobacterium binary vectors [ 150 , 151 ]. A variety of agroinoculation methods were developed during the last decade in order to examine the effect of transient gene expression, with applications ranging from monitoring of plant-pathogen interactions [ 152 ], subcellular localization [ 153 ], ectopic up-/down-regulation [ 12 ], in vivo analysis of plant promoters and transcription factors [ 154 , 155 ] to the production of recombinant proteins [ 156 , 157 ]. Efforts of several laboratories have resulted in the development of various methods for Agrobacterium tumefaciens –mediated gene transfer.…”