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New Imaging Method Enables Multiplexed Spatial Gene Expression Analysis in Plants


This article has been updated to correct that PHYTOMap implemented techniques from the spatially-resolved transcript amplicon readout mapping (STARMap) method. 

NEW YORK – Researchers from the Salk Institute for Biological Studies and their collaborators have developed a new fluorescence in situ hybridization method that enables multiplexed spatial gene expression analysis in plants.

Described in Nature Plants earlier this month, the approach, dubbed plant hybridization-based targeted observation of gene expression map (PHYTOMap), allows researchers to profile the expression of multiple genes within whole-mount plant tissue at single-cell resolution.

"Recently, the plant biology field has embraced single-cell transcriptomics," said Tatsuya Nobori, a postdoctoral researcher at the Salk Institute who is the lead author of the study. "This really accelerated the integration of genomics and cell biology."

Despite the growing popularity of single-cell studies in plant biology, Nobori said, the field still lacked a cost-effective and convenient spatial gene expression method to profile candidate marker genes in situ.

According to Nobori, one technical hurdle for applying single-cell and spatial biology techniques in plant cells is their cell wall, which needs to be permeabilized first during the experiment. In addition, plant samples tend to have high autofluorescence, especially in leaf tissue that contains chlorophyll, posing a challenge for imaging.

One traditional technique commonly used for spatial gene expression analysis in plants involves generating transgenic reporter lines, where researchers fuse genes encoding fluorescent proteins with the predicted promoter region to help visualize gene expression. However, this approach is "painstakingly slow," Nobori said, often taking several months, and can usually only analyze one gene per transgenic line.

Meanwhile, a number of commercially available spatial gene expression approaches have been applied to plants more recently, enabling researchers to analyze multiple genes at once. Still, in addition to being costly, those assays typically require tissue sectioning, meaning the native three-dimensional tissue structure is lost during the experiment, Nobori pointed out.

"That's why we thought that the plant biology community needs a spatial transcriptomic method that can work in 3D whole-mount tissue," he added.

Mechanistically, PHYTOMap functions similarly to spatially-resolved transcript amplicon readout mapping (STARMap), an in situ sequencing method developed by Karl Deisseroth’s lab at Stanford University. The method first replaces mRNA molecules with barcoded DNA probes, which are then amplified in situ prior to FISH, augmenting the signal-to-noise ratio. According to Nobori, the enhanced signal is essential for PHYTOMap to perform in whole-mount plant tissues, especially those with a large tissue volume or with high autofluorescence potential.

The expression signals are subsequently captured in situ using a confocal microscope. During each imaging round, four gene targets are detected, and multiplexing is achieved by sequential rounds of probing, imaging, and probe stripping, Nobori noted.

Additionally, the researchers developed a computational pipeline, which includes image registration, gene identification, cell segmentation, and single-cell gene expression analysis.

In their published proof-of-concept study, the researchers applied PHYTOMap in the model organism Arabidopsis thaliana and analyzed 28 target marker genes in a root tip. Overall, the results showed that the method could successfully pick out expression signals of validated marker genes in their expected cell type regions. The team also tested PHYTOMap in whole-mount Arabidopsis leaves, where the method successfully detected a housekeeping gene.

The study demonstrated that PHYTOMap can currently detect 50 genes in the same tissue. While the researchers did not "extensively" test PHYTOMap in other plants, Nobori said the technology can theoretically also be applied to other species.

In terms of the workflow, the method is "quite straightforward" to carry out, he said. The sample prep step for each experiment takes four to five days, including roughly 10 hours of total bench time. In the current study, each imaging round took three hours for one root tip and five hours for five root tips.

As for cost, Nobori said the current estimate for the protocol is about $80 per sample. Meanwhile, the initial reagent cost to set up PHYTOMap is approximately $2,700 for a 28-gene experiment.

"I'm very excited about [this method]," said David Jackson, a plant genetics professor at Cold Spring Harbor Laboratory who was not involved in the study. "This is one of the first methods in plants where you can look at many genes in the same sample, which is very exciting."

Although commercialized spatial transcriptomics assays, such as those from 10x Genomics, Vizgen, and Resolve Biosciences, have been used to study plant samples, one of the main challenges of these approaches is that they can be "extremely expensive," Jackson said, sometimes costing tens of thousands of dollars per experiment. In addition, some of these commercialized methods cannot achieve single-cell resolution in plants, he added.

Despite PHYTOMap's promises, Jackson said it remains to be seen how easily it can be replicated in other labs. "It does require quite a lot of technical expertise, both on the molecular biology side and also on the imaging and then computational side," he pointed out.

Because the Arabidopsis root tips used in the study can be "extremely thin," Jackson noted, one future direction would be to benchmark PHYTOMap in other plants, such as rice or maize, which have thicker root or shoot tissues. Similarly, since the authors only benchmarked PHYTOMap in wild-type tissues, he said another future step would be to evaluate how well the method can detect gene expression changes in plants under environmental pressure, such as heat or drought, or pathogen invasion.

Echoing Jackson's point, Nobori said one of the team's goals moving forward is to apply PHYTOMap to biological questions, such as plant-microbe interactions. "It's a really perfect method to map where those microbes are sitting in plants, and how the individual plant cells are responding to those microorganisms surrounding them," he noted.

In addition, he said the team plans to continue improving PHYTOMap. While the method's current multiplexing capability is around 50 genes, he believes it can be increased to hundreds or even more than 1,000 genes by optimizing the detection approach. Another direction is to make PHYTOMap multimodal, he said, enabling the technology to not only detect mRNAs but also other analytes, such as proteins, metabolites, or even microbes, at the same time.

The authors of the study have also applied for patents pertaining to PHYTOMap. "We don't have a plan to commercialize it right now," Nobori said. "But that is a possibility in the future."