Three-dimensional imaging of xylem at cell wall level through near field nano holotomography

Koddenberg, Tim; Greving, Imke; Hagemann, J.; Flenner, Silja; Krause, Andreas; Laipple, Daniel; Klein, Kim C.; Schmitt, Uwe; Schuster, Max; Wolf, Andreas; Seifert, Maria; Ludwig, Veronika; Funk, Stefan; Militz, Holger; Nopens, Martin

doi:10.1038/s41598-021-83885-8

Cited by 11 publications

(8 citation statements)

References 42 publications

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“…Of particular importance are the bordered pits at the lateral scalariform wall of vessels and tracheids and the half-bordered pits at the boundaries with RP and VACs. They form an interface between apoplastic xylem elements and the symplast, constituting a three-dimensional network in the plant trunk that extend to the shoot tips (Kedrov, 2013;Sano et al, 2013;Donaldson et Gao et al, 2020;Zhang et al, 2020;Kaack et al, 2021;Koddenberg et al, 2021;).…”

Section: Discussionmentioning

confidence: 99%

Trunk anatomy of asymptomatic and symptomatic grapevines provides insights into degradation patterns of wood tissues caused by Esca-associated pathogens

Kassemeyer

KLUGE

BIELER³

et al. 2022

Phytopathol. Mediterr.

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Wood colonizing fungi are specialists that exploit the lignocellulose of cell wall components in host wood cylinders as a carbon sources. Some of these specialized fungi, including Fomitiporia mediterranea (Fmed) and Phaeomoniella chlamydospora (Pch), cause the disease Esca of grapevine. This disease complex includes grapevine leaf stripe disease (GLSD) of canopies and white rot and black wood streaking in trunks. The present study gained insights into the activity of Esca pathogens in host xylem of the trunk tissues at an anatomical level. Lesions with white rot and brown wood streaking were microscopically analyzed, and the structures of affected tissues were compared with intact xylem. In trunks with white rot, demarcation zones separated intact tissues from the lesions. Immediately adjacent to the demarcation zones, cell wall decomposition initiated in the xylem. At this initial stage, cavities appeared in the secondary cell walls of libriform fibres, which expanded and closely resembled the degradation pattern of soft rot. In the advanced stage, the fibre cell walls were completely decomposed, and the vessels were attacked with a degradation pattern similar to white rot. Only remnants of the xylem elements remained, forming amorphous matrices. These decomposition patterns occurred in field samples and in wood cores artificially infected with Fmed. The obvious compartmentalization of the tissue affected by Fmed indicated a defense reaction in the xylem, according to the CODIT model. In contrast, brown wood streaking affected only small groups of vessels, adjacent libriform fibres and parenchyma. Dark inclusions in cells and tyloses in vessels indicate a defense reaction against the pathogens causing brown wood streaking. Artificial inoculation of sterile wood cores with Pch confirmed the contribution of this pathogen to brown wood streaking. This research provides insights into the structural and functional anatomy of intact and infected tissues of grapevines, which clarify the etiology of Esca, and provide new knowledge for developing new approaches to control of this disease complex.

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Section: Discussionmentioning

confidence: 99%

Trunk anatomy of asymptomatic and symptomatic grapevines provides insights into degradation patterns of wood tissues caused by Esca-associated pathogens

Kassemeyer

KLUGE

BIELER³

et al. 2022

Phytopathol. Mediterr.

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“…Lower currents increase the amount of time needed to prepare a sample. Furthermore, the gallium ions used for thinning can compromise the experiment by implanting within the sample during milling (Koddenberg et al ., 2021), and high-energy gallium ions damage the exposed surfaces of the lamellae (Eder et al ., 2021; Kelley et al ., 2013). These damaged faces lower achievable signal-to-noise ratio.…”

Section: Mainmentioning

confidence: 99%

A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals

Martynowycz

Shiriaeva

Clabbers

et al. 2022

Preprint

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Crystallization of membrane proteins, such as G protein-coupled receptors (GPCRs), is challenging and frequently requires the use of lipidic cubic phase (LCP) crystallization methods. These typically yield crystals that are too small for synchrotron X-ray crystallography, but ideally suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, the viscous nature of LCP makes sample preparation challenging. The LCP layer is often too thick for transmission electron microscopy (TEM), and crystals buried in LCP cannot be identified topologically using a focused ion-beam and scanning electron microscope (FIB/SEM). Therefore, the LCP needs to either be converted to the sponge phase or entirely removed from the path of the ion-beam to allow identification and milling of these crystals. Unfortunately, conversion of the LCP to sponge phase can also deteriorate the sample. Methods that avoid LCP conversion are needed. Here, we employ a novel approach using an integrated fluorescence light microscope (iFLM) inside of a FIB/SEM to identify fluorescently labelled crystals embedded deep in a thick LCP layer. The crystals are then targeted using fluorescence microscopy and unconverted LCP is removed directly using a plasma focused ion beam (pFIB). To assess the optimal ion source to prepare biological lamellae, we first characterized the four available gas sources on standard crystals of the serine protease, proteinase K. However, lamellae prepared using either argon and xenon produced the highest quality data and structures. Fluorescently labelled crystals of the human adenosine receptor embedded in thick LCP were placed directly onto EM grids without conversion to the sponge phase. Buried microcrystals were identified using iFLM, and deep lamellae were created using the xenon beam. Continuous rotation MicroED data were collected from the exposed crystalline lamella and the structure was determined using a single crystal. This study outlines a robust approach to identifying and milling LCP grown membrane protein crystals for MicroED using single microcrystals, and demonstrates plasma ion-beam milling as a powerful tool for preparing biological lamellae.

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“…Lower currents increase the amount of time needed to prepare a sample. Furthermore, the gallium ions used for thinning can compromise the experiment by implanting within the sample during milling 20 , and high-energy gallium ions damage the exposed surfaces of the lamellae 21 , 22 . These damaged surfaces lower the achievable signal-to-noise ratio.…”

Section: Introductionmentioning

confidence: 99%

A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals

et al. 2023

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Crystallizing G protein-coupled receptors (GPCRs) in lipidic cubic phase (LCP) often yields crystals suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, sample preparation is challenging. Embedded crystals cannot be targeted topologically. Here, we use an integrated fluorescence light microscope (iFLM) inside of a focused ion beam and scanning electron microscope (FIB-SEM) to identify fluorescently labeled GPCR crystals. Crystals are targeted using the iFLM and LCP is milled using a plasma focused ion beam (pFIB). The optimal ion source for preparing biological lamellae is identified using standard crystals of proteinase K. Lamellae prepared using either argon or xenon produced the highest quality data and structures. MicroED data are collected from the milled lamellae and the structures are determined. This study outlines a robust approach to identify and mill membrane protein crystals for MicroED and demonstrates plasma ion-beam milling is a powerful tool for preparing biological lamellae.

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Three-dimensional imaging of xylem at cell wall level through near field nano holotomography

Cited by 11 publications

References 42 publications

Trunk anatomy of asymptomatic and symptomatic grapevines provides insights into degradation patterns of wood tissues caused by Esca-associated pathogens

Trunk anatomy of asymptomatic and symptomatic grapevines provides insights into degradation patterns of wood tissues caused by Esca-associated pathogens

A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals

A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals

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