Laser microdissection of semi-thin sections from plastic-embedded tissue for studying plant–organism developmental processes at single-cell resolution

Klink, Vincent P.; Thibaudeau, Giselle

doi:10.1080/17429145.2013.879677

Cited by 5 publications

(6 citation statements)

References 78 publications

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“…It is anticipated that the nonaxenic root transformation approach presented here would be useful for studies involving plant-organism interactions or other aspects of root biology in G. hirsutum (Klink & Thibaudeau 2014). Prior studies in other agriculturally relevant plant systems have already shown that this system is capable of producing tens of thousands of plants that can be easily maintained at one time for experimental study (Matthews et al 2013;Pant, Matsye, et al 2014).…”

Section: Compatibility For Studies Involving Root-dwelling Organismsmentioning

confidence: 99%

“…The system presented here differs from other hairy root-based systems developed in G. hirsutum in that, by using eGFP as a reporter system, the experiments can be performed under nonaxenic conditions throughout the study (Triplett et al 2008;Kim et al 2011;Matsye et al 2012;Pant, Matsye, et al 2014;. Furthermore, the platform is Gateway® compatible which simplifies the isolation and study of hundreds to thousands of genes identified in bioinformatics analyses (Matsye et al 2012;Matthews et al 2013;Pant, Matsye, et al 2014;Klink & Thibaudeau 2014). This facet of the genetic engineering platform greatly lessens the amount of technical expertise that is required for successful plant transformation.…”

Section: Introductionmentioning

confidence: 99%

“…This facet of the genetic engineering platform greatly lessens the amount of technical expertise that is required for successful plant transformation. Notably, this work is the compliment to Klink and Thibaudeau (2014) for the analysis of G. hirsutum root interaction research.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

A plant transformation system designed for high throughput genomics inGossypium hirsutumto study root–organism interactions

Pant

McNeece

Sharma

et al. 2015

Journal of Plant Interactions

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The study of biological processes has been aided greatly by the development of procedures to identify the large numbers of associated genes. However, the ability to study the identified genes experimentally is often impeded by the absence of technologies to perform such functional analyses. Here, a nonaxenic plant transformation system has been developed in Gossypium hirsutum (cotton) for the study of genes associated with root functions and root-organism interactions. The plant transformation system is compatible with modern high throughput plant transformation goals and the processing of large numbers of genes intended for the study of root function in G. hirsutum.

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Section: Compatibility For Studies Involving Root-dwelling Organismsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A plant transformation system designed for high throughput genomics inGossypium hirsutumto study root–organism interactions

Pant

McNeece

Sharma

et al. 2015

Journal of Plant Interactions

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“…While there are variations in LMD design from different manufacturers, tissues are generally fixed and sectioned using common histological techniques and placed on specialized microscopy slides or plates. Depending on the system of study, wax or plastic can be used to embed tissues and preserve RNA ( Inada and Wildermuth, 2005 ; Klink and Thibaudeau, 2014 ). Once fixed, the samples are then visualized using light or fluorescence microscopy and individual cells and tissues are selected and excised with a laser and collected for downstream molecular analysis ( Schiebold et al, 2011 ; Gautam and Sarkar, 2015 ).…”

Section: Laser Dissection Of Host–pathogen Interactionsmentioning

confidence: 99%

Integrating Large-Scale Data and RNA Technology to Protect Crops from Fungal Pathogens

et al. 2016

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With a rapidly growing human population it is expected that plant science researchers and the agricultural community will need to increase food productivity using less arable land. This challenge is complicated by fungal pathogens and diseases, many of which can severely impact crop yield. Current measures to control fungal pathogens are either ineffective or have adverse effects on the agricultural enterprise. Thus, developing new strategies through research innovation to protect plants from pathogenic fungi is necessary to overcome these hurdles. RNA sequencing technologies are increasing our understanding of the underlying genes and gene regulatory networks mediating disease outcomes. The application of invigorating next generation sequencing strategies to study plant–pathogen interactions has and will provide unprecedented insight into the complex patterns of gene activity responsible for crop protection. However, questions remain about how biological processes in both the pathogen and the host are specified in space directly at the site of infection and over the infection period. The integration of cutting edge molecular and computational tools will provide plant scientists with the arsenal required to identify genes and molecules that play a role in plant protection. Large scale RNA sequence data can then be used to protect plants by targeting genes essential for pathogen viability in the production of stably transformed lines expressing RNA interference molecules, or through foliar applications of double stranded RNA.

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“…To improve the uniformity and quality of analyzed samples, enrichment in purified material originating from the syncytial protoplasts is desirable. Such localized specificity of the sample can be achieved by microaspiration of the syncytial cytoplasm [5] or laser-capture microdissection (LCM) of syncytia from infected root sections [29][30][31][32]. In particular, given its relative ease and reliability, an increasing number of successful biological applications of LCM has allowed isolation of RNA, DNA, proteins, and metabolites from heterogeneous populations of cells [33,34].…”

Section: Introductionmentioning

confidence: 99%

Profiling the Proteome of Cyst Nematode-Induced Syncytia on Tomato Roots

Filipecki

Żurczak

Matuszkiewicz

et al. 2021

IJMS

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Cyst nematodes are important herbivorous pests in agriculture that obtain nutrients through specialized root structures termed syncytia. Syncytium initiation, development, and functioning are a research focus because syncytia are the primary interface for molecular interactions between the host plant and parasite. The small size and complex development (over approximately two weeks) of syncytia hinder precise analyses, therefore most studies have analyzed the transcriptome of infested whole-root systems or syncytia-containing root segments. Here, we describe an effective procedure to microdissect syncytia induced by Globodera rostochiensis from tomato roots and to analyze the syncytial proteome using mass spectrometry. As little as 15 mm2 of 10-µm-thick sections dissected from 30 syncytia enabled the identification of 100–200 proteins in each sample, indicating that mass-spectrometric methods currently in use achieved acceptable sensitivity for proteome profiling of microscopic samples of plant tissues (approximately 100 µg). Among the identified proteins, 48 were specifically detected in syncytia and 7 in uninfected roots. The occurrence of approximately 50% of these proteins in syncytia was not correlated with transcript abundance estimated by quantitative reverse-transcription PCR analysis. The functional categories of these proteins confirmed that protein turnover, stress responses, and intracellular trafficking are important components of the proteome dynamics of developing syncytia.

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Laser microdissection of semi-thin sections from plastic-embedded tissue for studying plant–organism developmental processes at single-cell resolution

Cited by 5 publications

References 78 publications

A plant transformation system designed for high throughput genomics inGossypium hirsutumto study root–organism interactions

A plant transformation system designed for high throughput genomics inGossypium hirsutumto study root–organism interactions

Integrating Large-Scale Data and RNA Technology to Protect Crops from Fungal Pathogens

Profiling the Proteome of Cyst Nematode-Induced Syncytia on Tomato Roots

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