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Int. J. Mol. Sci. 2018, 19(2), 429; https://doi.org/10.3390/ijms19020429

A Phenotyping Method of Giant Cells from Root-Knot Nematode Feeding Sites by Confocal Microscopy Highlights a Role for CHITINASE-LIKE 1 in Arabidopsis

1
Facultad de Ciencias Ambientales y Bioquímica, Universidad de Castilla-La Mancha, Área de Fisiología Vegetal, Avda, Carlos III, s/n, 45071 Toledo, Spain
2
Centro de Biotecnología y Genómica de Plantas (UPM-INIA), Pozuelo de Alarcón, 28223 Madrid, Spain
3
Laboratory of Plant Physiology and Molecular Genetics, Université libre de Bruxelles, Campus Plaine CP 242, Bd du Triomphe, 1050 Brussels, Belgium
4
Facultad de Ciencias Ambientales y Bioquímica, Universidad de Castilla-La Mancha, Área de Genética, Avda, Carlos III, s/n, 45071 Toledo, Spain
*
Author to whom correspondence should be addressed.
Received: 21 December 2017 / Revised: 19 January 2018 / Accepted: 26 January 2018 / Published: 1 February 2018
(This article belongs to the Special Issue Plant Defense Genes Against Biotic Stresses)
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Abstract

Most effective nematicides for the control of root-knot nematodes are banned, which demands a better understanding of the plant-nematode interaction. Understanding how gene expression in the nematode-feeding sites relates to morphological features may assist a better characterization of the interaction. However, nematode-induced galls resulting from cell-proliferation and hypertrophy hinders such observation, which would require tissue sectioning or clearing. We demonstrate that a method based on the green auto-fluorescence produced by glutaraldehyde and the tissue-clearing properties of benzyl-alcohol/benzyl-benzoate preserves the structure of the nematode-feeding sites and the plant-nematode interface with unprecedented resolution quality. This allowed us to obtain detailed measurements of the giant cells’ area in an Arabidopsis line overexpressing CHITINASE-LIKE-1 (CTL1) from optical sections by confocal microscopy, assigning a role for CTL1 and adding essential data to the scarce information of the role of gene repression in giant cells. Furthermore, subcellular structures and features of the nematodes body and tissues from thick organs formed after different biotic interactions, i.e., galls, syncytia, and nodules, were clearly distinguished without embedding or sectioning in different plant species (Arabidopsis, cucumber or Medicago). The combination of this method with molecular studies will be valuable for a better understanding of the plant-biotic interactions. View Full-Text
Keywords: BABB; clearing; confocal microscopy; CTL1; giant cells; Meloidogyne spp.; nodules; syncytia BABB; clearing; confocal microscopy; CTL1; giant cells; Meloidogyne spp.; nodules; syncytia
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Cabrera, J.; Olmo, R.; Ruiz-Ferrer, V.; Abreu, I.; Hermans, C.; Martinez-Argudo, I.; Fenoll, C.; Escobar, C. A Phenotyping Method of Giant Cells from Root-Knot Nematode Feeding Sites by Confocal Microscopy Highlights a Role for CHITINASE-LIKE 1 in Arabidopsis. Int. J. Mol. Sci. 2018, 19, 429.

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