Phenotyping nematode feeding sites: three‐dimensional reconstruction and volumetric measurements of giant cells induced by root‐knot nematodes in Arabidopsis

Abstract

SummaryThe control of plant parasitic nematodes is an increasing problem. A key process during the infection is the induction of specialized nourishing cells, called giant cells (GCs), in roots. Understanding the function of genes required for GC development is crucial to identify targets for new control strategies. We propose a standardized method for GC phenotyping in different plant genotypes, like those with modified genes essential for GC development.The method combines images obtained by bright-field microscopy from the complete serial sectioning of galls with TRAKEM2, specialized three-dimensional (3D) reconstruction software for biological structures.The volumes and shapes from 162 3D models of individual GCs induced by Meloidogyne javanica in Arabidopsis were analyzed for the first time along their life cycle. A high correlation between the combined volume of all GCs within a gall and the total area occupied by all the GCs in the section/s where they show maximum expansion, and a proof of concept from two Arabidopsis transgenic lines (J0121 ≫ DTA and J0121 ≫ GFP) demonstrate the reliability of the method.We phenotyped GCs and developed a reliable simplified method based on a two-dimensional (2D) parameter for comparison of GCs from different Arabidopsis genotypes, which is also applicable to galls from different plant species and in different growing conditions, as thickness/transparency is not a restriction.

Application of Nuclear Volume Measurements to Comprehend the Cell Cycle in Root-Knot Nematode-Induced Giant Cells

“…Ours and other studies have described methodologies to visualize nuclei in vivo and/or in fixed GCs in nematode feeding sites using confocal microscopy but none of these studies report GC nuclear volumetric measurements (Vieira et al, 2012, 2013, 2014; Dinh et al, 2014; Hasegawa et al, 2016; Coelho et al, 2017). Different methods have been applied to measure nematode-induced GC area to deduce corresponding volumes (Vieira et al, 2013, 2014; Cabrera et al, 2015) and purely their nuclear area (Vieira et al, 2013, 2014), but there are no reports on GC nuclear volumes. Surface measurements of GC nuclei were reported by Vieira et al (2013, 2014) using DAPI-stained plastic embedded and sectioned galls.…”

The Plant Cell Cycle Machinery: Usurped and Modulated by Plant-Parasitic Nematodes

“…Recently a protocol has been developed based on classical histological tissue sectioning and 3D volumetric measurement analysis (Cabrera et al, 2015) which is not limited by specimen dimensions and that allows 3D morphological analysis of much bigger and less transparent galls. This approach is easy implementable in many laboratories and will be useful to gather 3D data from galls and GCs from anatomically more complex crops.…”

Long-Term In Vitro System for Maintenance and Amplification of Root-Knot Nematodes in Cucumis sativus Roots

“…In contrast, J2s from the described aseptic culture were in the optimum infectivity state that did not vary much in different batches. This allowed the use of a reduced nematode inoculum (10 nematodes per plant) to avoid undesired root damage (Cabrera et al, 2014, 2015). Another advantage of the method herein described is that it allows for three independent biological replicates by infecting plants 4 days apart with J2 hatched from the same egg mass pool from three independent hatchings.…”

“…Moreover, J2s from these cultures can be used directly after a simple hatching step in aseptic conditions to infect plants for experiments with different in vitro grown plant species. Successful experiments have been performed with this procedure on different genotypes of Arabidopsis , tobacco, and tomato (Barcala et al, 2010; Escobar et al, 2010; Portillo et al, 2013; Cabrera et al, 2014, 2015, 2016). …”