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ARS Home » Plains Area » Fargo, North Dakota » Edward T. Schafer Agricultural Research Center » Cereal Crops Research » Research » Publications at this Location » Publication #409535

Research Project: Host-Pathogen Interactions Affecting Wheat and Barley

Location: Cereal Crops Research

Title: Unleashing the secrets of plant-fungal interactions using a transformation-free confocal staining technique that supports AI-assisted quantitative analysis

Author
item NELSON, ASHLEY - North Dakota State University
item KARIYAWASAM, GAYAN - North Dakota State University
item Wyatt, Nathan
item LI, JING - North Dakota State University
item HAUEISEN, JANINE - Max Planck Institute For Evolutionary Biology
item STUKENBROCK, EVA - Max Planck Institute For Evolutionary Biology
item BOROWICZ, PAWEL - North Dakota State University
item Friesen, Timothy

Submitted to: bioRxiv
Publication Type: Pre-print Publication
Publication Acceptance Date: 10/6/2023
Publication Date: 10/6/2023
Citation: Nelson, A., Kariyawasam, G., Wyatt, N.A., Li, J., Haueisen, J., Stukenbrock, E., Borowicz, P., Friesen, T.L. 2023. Unleashing the secrets of plant-fungal interactions using a transformation-free confocal staining technique that supports AI-assisted quantitative analysis. bioRxiv. https://doi.org/10.1101/2023.10.04.560942.
DOI: https://doi.org/10.1101/2023.10.04.560942

Interpretive Summary:

Technical Abstract: Laser scanning confocal microscopy’s ability to generate high-contrast 3D images has become essential to studying plant-fungal interactions. Techniques such as visualization of native fluorescence, fluorescent protein tagging of microbes, GFP/RFP-fusion proteins, and fluorescent labelling of plant and fungal proteins have been widely used to aid in these investigations. Use of fluorescent proteins have several pitfalls including variability of expression in planta and the requirement of gene transformation. Here we used the unlabeled pathogens Parastagonospora nodorum, Pyrenophora teres f. teres, and Cercospora beticola infecting wheat, barley, and sugar beet respectively, to show the utility of a staining and imaging technique that uses propidium iodide (PI) and wheat germ agglutinin labeled with fluorescein isothiocyanate (WGA-FITC) to visualize fungal colonization of plants. This method relies on the use of KOH to remove the cutin layer of the leaf, increasing its permeability. This permeability allows the staining solution to penetrate and efficiently bind to its targets, resulting in a consistent visualization of cellular structures. We have also used this staining technique in conjunction with machine learning to analyze fungal volume (biomass), which indicates the fitness of the pathogen in planta, as well as quantifying nuclear breakdown, an early indicator of programmed cell death (PCD).This technique is simple to use, robust, consistent across host species, and can be applied to any plant-fungal interaction. Therefore, this technique can be used to characterize model systems as well as non-model interactions where transformation is not routine.