Micro-colonies developed in the rhizosphere . Since E. As a pathogenic bacteria, E. When using transparent soil, there is a potential problem of moving the bacteria during saturation of the substrate in preparation for imaging but this should not affect attached bacteria such as E. Saturation is, however, a potential limitation of the method if studying microbes that are not attached to surfaces because it is likely that these would be moved during saturation.
In summary, our results show that transparent soil is ideal for imaging studies of certain plant-microbe interactions in situ at the microscopic level. Soil microbes provide numerous important services  and their interactions with plants enhance the supply of nutrients, for example by nodulation  or by biological fertilization . The transfer of human pathogens in the food chain  and spread of crop diseases  also involve complex plant-microbe interactions. The use of transparent soil will facilitate quantitative imaging of the dynamics of in situ root-microbe interactions using high resolution imaging with fluorescence for detecting microorganisms expressing fluorescent proteins Fig.
For plant genetics and crop breeding, transparent soil could be integrated with high-throughput screening systems for root traits  that may improve nutrient acquisition and reduce the need for fertilizers . Overall, this approach presents new opportunities to unravel the complex processes of plant-soil interactions in situ and in vivo and holds promise for a wide range of applications to aid the understanding of important underlying relationships that underpin the sustainability of our ecosystems.
Nafion was from Ion Power Inc. Acid NR50 and precursor R1 forms were used. After cooling, the particles were washed several times with dH 2 O. The particles were rinsed again multiple times with fresh dH 2 O . To titrate the particles with mineral ions, stock solutions of MSR media were used to immerse the particles. This was repeated until the pH was neutral and stable. Before use, the particles were autoclaved in dH 2 O for sterilisation. The straightness of the line for each image was used as an indicator of the light path distortion by refraction.
The thresholded image was skeletonized and a bounding box around the line was created. Nutrient-titrated Nafion particles were also tested in this way, but with a larger range of sorbitol concentrations. Saturated samples were placed on ceramic plates in glass funnels, which were connected to hanging water columns. Different suctions were achieved by moving the water level in the water column to a specific height.
At each pressure, the water content of the sample was allowed to equilibrate and the mass was recorded to allow calculation of volumetric water content. Data on water retention in vermiculite and sand from other studies were used for comparison with our data on water retention in transparent soil  , . Exchangeable cations were extracted using the ammonium acetate method  and cation exchange capacity was quantified by subsequent ICP-MS analysis.
To measure anion exchange capacity, sorbed chloride ions were exchanged with nitrate ions and exchange capacity was determined by measuring the extracted chloride ions . Arabidopsis thaliana expressing 35S:LTI6b- EGFP constitutively expressed enhanced green fluorescent protein targeted to the plasma membrane , in the C24 background originally obtained from Dr. Haseloff, University of Cambridge, UK  and auxin reporter lines  were used for confocal microscopy.
Nicotiana benthamiana tobacco and Latuca sativa lettuce, var. The substrates used for analysing plant growth were 1. The soil was sieved to 3 mm and packed to a density of 1. Horticultural grit sand Gem, UK , with a dry bulk density of 1. Transparent soil, prepared as described below and packed to a density of 1. All plants were excavated, the roots were washed and they were mounted onto acetate sheets for scanning using a flatbed scanner Epson expression XL.
Imaging was carried out after 5 days after sowing. The method used for bacteria-plant interactions allowed colonization of the roots to develop from infected seedlings, rather than from the addition of the inoculum directly to the substrate or the more mature roots. For OPT imaging the samples were prepared in glass cylindrical specimen tubes 2. Duration of growth was dependent on plant species but in general, imaging was performed before the roots reached the base of the tube. Tobacco plants used for OPT were imaged 10 days after sowing. Arabidopsis plants used for confocal imaging were imaged 10—14 days after sowing.
The stage and camera were controlled by software also built in-house, allowing control of the number of images acquired for each sample. The projection images were reconstructed to produce 3D data using a filtered backprojection algorithm with the Iradon function in Matlab The MathWorks, Inc. For CLSM, plants were grown in purpose-built chambers, constructed using a microscope slide and long cover glass with a 4 mm spacer between them on 3 sides and an opening at the top.
The spacer was glued to the slide and cover glass using Araldite glass and ceramic adhesive Huntsman International. The chambers were covered with aluminium foil on the outside during growth to exclude light from the roots. Foil was removed immediately before imaging. The refractive index of the solution matches the refractive index of the Nafion particles used here to provide complete transparency in the substrate.
Sigmaplot 12 Syststat Software, Inc. Image analysis was carried out using Mevislab  and Fiji Software .
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Root tracking used an algorithm by Friman et al . Skeletonization and edge detection was carried out using the standard Mevislab algorithms developed respectively by Milo Hindennach and Olaf Konrad and Wolf Spindler. Snapshots of volume renderings of confocal scans of Arabidopsis thaliana roots expressing GFP in plasma membranes grey in transparent soil with sulphorhdamine-B-dyed particles orange.
Lateral root emerging from primary root.
Section of primary root and root hairs in contact with Nafion particle. In situ 3D image of branched Arabidopsis thaliana roots expressing GFP in plasma membranes green in transparent soil with sulphorhdamine-B-dyed particles red. In situ 3D image of Arabidopsis thaliana root with emrging lateral root expressing GFP in plasma membranes green in transparent soil with sulphorhdamine-B-dyed particles orange. In situ 3D image of Arabidopsis thaliana root with root hairs expressing GFP in plasma membranes green with Nafion particle of transparent soil orange.
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Yossef A. Performed the experiments: HD. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Understanding of soil processes is essential for addressing the global issues of food security, disease transmission and climate change. Introduction The ability of plants and microorganisms to successfully exploit soil resources underpins the survival of all terrestrial life. Results and Discussion Making Soils Transparent using Refractive Index Matching At the boundary of two transparent materials with different refractive indices, the path of light is distorted through refraction.
Interactions of beneficial and detrimental root-colonizing filamentous microbes with plant hosts
Mimicking Physical and Chemical Properties of Soil We have also sought to mimic physical and chemical properties important for supporting plant and microbial growth in soils in the transparent soil system. Root Growth in Transparent Soil Transparent soil can be used for a large range of other applications. Figure 2. Imaging roots and microorganisms in transparent soil using OPT and confocal microscopy. Application of Transparent Soil to the Study of Root Bacteria Interactions We have applied transparent soil to study the mechanisms of transmission of food-borne human pathogens on fresh produce plants using GFP-labelled Escherichia coli OH7.
New Opportunities for Plant Sciences Soil microbes provide numerous important services  and their interactions with plants enhance the supply of nutrients, for example by nodulation  or by biological fertilization . Analysis of Plant Growth in Different Substrates The substrates used for analysing plant growth were 1.
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