Characterizing virus-induced gene silencing at the cellular level with in situ multimodal imaging

Abstract

BackgroundReverse genetic strategies, such as virus-induced gene silencing, are powerful techniques to study gene function. Currently, there are few tools to study the spatial dependence of the consequences of gene silencing at the cellular level.ResultsWe report the use of multimodal Raman and mass spectrometry imaging to study the cellular-level biochemical changes that occur from silencing the phytoene desaturase (pds) gene using a Foxtail mosaic virus (FoMV) vector in maize leaves. The multimodal imaging method allows the localized carotenoid distribution to be measured and reveals differences lost in the spatial average when analyzing a carotenoid extraction of the whole leaf. The nature of the Raman and mass spectrometry signals are complementary: silencing pds reduces the downstream carotenoid Raman signal and increases the phytoene mass spectrometry signal.ConclusionsBoth Raman and mass spectrometry imaging show that the biochemical changes from FoMV-pds silencing occur with a mosaic spatial pattern at the cellular level, and the Raman images show carotenoid expression was reduced at discrete locations but not eliminated. The data indicate the multimodal imaging method has great utility to study the biochemical changes that result from gene silencing at the cellular spatial level of expression in many plant tissues including the stem and leaf. Our demonstrated method is the first to spatially characterize the biochemical changes as a result of VIGS at the cellular level using commonly available instrumentation.Electronic supplementary materialThe online version of this article (10.1186/s13007-018-0306-7) contains supplementary material, which is available to authorized users.

High mass resolution, spatial metabolite mapping enhances the current plant gene and pathway discovery toolbox

“…Instead, the co‐existence of multiple phenotypes in the same sample facilitates the quantitative comparison of different phenotypes, because the intersample variations and batch effects are eliminated. Burkhow and colleagues (Burkhow et al ., 2018) spatially characterised the biochemical changes in maize leaves following VIGS‐based silencing using multimodal Raman and MALDI imaging. In a similar manner, by combining VIGS, MALDI imaging and LC‐MS we studied GAME31, a 2‐oxoglutarate dependent dioxygenase, catalysing the hydroxylation of α ‐tomatine to hydroxytomatine in the tomato esculeoside A biosynthetic pathway (Cárdenas et al ., 2019) (Fig.…”

Perspective on Multimodal Imaging Techniques Coupling Mass Spectrometry and Vibrational Spectroscopy: Picturing the Best of Both Worlds

“…Raman described the spatial distribution of carotenoids, but due to the lack of Raman signal to detect phytoenes, SALDI was chosen to monitor phytoene accumulation when they silenced the chosen gene. 6 In another study, combined information from SERS and LA-ICP-MS demonstrated that hybrid nanoparticles penetrate fibroblast cells differently from standard silica nanoparticles. 14 The SERS spectra reflected the interaction of several amino acids of the side chains of proteins with the nanoparticles’ surface, while LA-ICP images revealed the cellular uptake of silica nanoparticles with a silver core by monitoring the stable isotope 107 Ag.…”

“…MSI and VSI can be combined to overcome the limitations of one technique and complementing it by the advantages of the other. This combination has been applied in various imaging studies (as illustrated in Table S1 in the Supporting Information): animal brains (mouse, ,, rat, , and hamster), cell cultures, , bacterial colonies, ,, liver metabolism, , cancerous tissues, ,, fingermarks, and even plants (perennial grass and maize leaves). Label-free multimodal imaging is a new holistic approach which will impact analytical chemistry, especially the “omics” sciences, for two main reasons: (1) the imaging techniques provide complementary information that identifies molecules and validates results and (2) the spatial and spectral information have boosted value.…”

“…monitored the conversion of phytoenes to carotenoids directly in maize leaves and how the rate of this conversion changed when they silenced one of the genes involved in the biosynthesis. Raman described the spatial distribution of carotenoids, but due to the lack of Raman signal to detect phytoenes, SALDI was chosen to monitor phytoene accumulation when they silenced the chosen gene . In another study, combined information from SERS and LA-ICP-MS demonstrated that hybrid nanoparticles penetrate fibroblast cells differently from standard silica nanoparticles .…”

“…However, one picture might not be enough to fully characterize complex biosystems. That is why multimodal imaging platforms have emerged in the past decade as a coordinated combination of multiple imaging techniques to find solutions in fields as diverse as plant-based renewable energy, microbiology, clinical medicine, and forensics. − Multimodal imaging provides a set of information which characterizes biological samples from two aspects: quantitative and qualitative molecular information and molecular distribution at both high and low spatial resolution. The coupling of imaging modalities started with optical microscopy techniques such as classical histopathology, fluorescence imaging, and immunohistochemistry being combined with molecular imaging techniques such as infrared or Raman spectroscopy imaging and mass spectrometry imaging (MSI). ,,− The classical histology techniques have a major limitation which impedes comprehensive analysis of biological samples: they use labels for targeted imaging. , This approach involves monitoring a reduced number of compounds (usually two or three proteins) because the chemical stains, immunohistochemical tags, or other labels used for imaging are limited.…”