Semiautomated confocal imaging of fungal pathogenesis on plants: Microscopic analysis of macroscopic specimens

Abstract

Studying phenotypic variation in plant pathogenesis provides fundamental information about the nature of disease resistance. Cellular mechanisms that alter pathogenesis can be elucidated with confocal microscopy, but systematic phenotyping platforms—from sample processing to image analysis—to investigate this do not exist. We have developed a platform for 3D phenotyping of cellular features underlying variation in disease development by fluorescence-specific resolution of host and pathogen interactions across time (4D). A confocal microscopy phenotyping platform compatible with different maize-fungal pathosystems (fungi: Setosphaeria turcica, Cochliobolus heterostrophus, and Cercospora zeae-maydis) was developed. Protocols and techniques were standardized for sample fixation, optical clearing, species-specific combinatorial fluorescence staining, multi-sample imaging, and image processing for investigation at the macro scale. The sample preparation methods presented here overcome challenges to fluorescence imaging such as specimen thickness and topography as well as physiological characteristics of the samples such as tissue autofluorescence and presence of cuticle. The resulting imaging techniques provide interesting qualitative and quantification information not possible with conventional light or electron 2D imaging.

Identification of Loci That Confer Resistance to Bacterial and Fungal Diseases of Maize

“…Despite the differences between fungal and bacterial pathogens, some of the pathogens can infect the same tissue types, specifically the vasculature. SCLB and GLS are non-vascular diseases ( Beckman and Payne 1982 ; Minker et al 2018 ), while GW and NCLB are vascular diseases ( Minker et al 2018 ; Mbofung et al 2016 ). Only one vascular/vascular (NCLB and GW) disease correlation combination was identified.…”

Shedding Light on Penetration of Cereal Host Stomata by Wheat Stem Rust Using Improved Methodology

“…The need of highly sensitive and less disruptive detection method for fungal pathogens, especially those that are recalcitrant to transformation, in order to characterize the early/limited spatial and temporal development of substomatal structures, made it necessary to develop an improved method preserving the sample integrity for in-planta fungal staining. 3D phenotyping of three fungal pathogens on maize was previously described using two photon microscopy 25 and fungal volume assessment was reported for mycorrhizal fungi 26 . Here we report on the development of an improved, cost effective and rapid method of fungal WGA-AF based staining, visualization, and bio-volume analysis of in-planta pathogens that works irrespective of their life style, biotroph or necrotroph, or their recalcitrance to transformation.…”

Bioimaging structural signatures of the oomycete pathogen Sclerospora graminicola in pearl millet using different microscopic techniques

“…Recently, various techniques have been improved by Minker et al . 38 for semi-automated confocal imaging of fungal pathogens on plants. Techniques and methods were optimized for sample fixation, optical clearing, species-specific fluorescence staining and image processing with reference to pathogenesis in maize tissues by the fungal pathogens Exserohilum turcicum, Bipolaris maydis , and Cercospora zeae-maydis 38,39 .…”

“…Techniques and methods were optimized for sample fixation, optical clearing, species-specific fluorescence staining and image processing with reference to pathogenesis in maize tissues by the fungal pathogens Exserohilum turcicum, Bipolaris maydis , and Cercospora zeae-maydis 38,39 . CLSM is a new advancement in light microscopy, which mostly explores the latest laser, imaging and computational technologies that enable biologists to vizualize a distinctive form of cell and/or subcellular components 38 . The blur-free images of thick specimens can also be obtained by using computer tools and fluorescent light reflection at various depths.…”