Abstract
Root-knot nematodes induce galls that contain giant-feeding cells harboring multiple enlarged nuclei within the roots of host plants. It is recognized that the cell cycle plays an essential role in the set-up of a peculiar nuclear organization that seemingly steers nematode feeding site induction and development. Functional studies of a large set of cell cycle genes in transgenic lines of the model host Arabidopsis thaliana have contributed to better understand the role of the cell cycle components and their implication in the establishment of functional galls. Mitotic activity mainly occurs during the initial stages of gall development and is followed by an intense endoreduplication phase imperative to produce giant-feeding cells, essential to form vigorous galls. Transgenic lines overexpressing particular cell cycle genes can provoke severe nuclei phenotype changes mainly at later stages of feeding site development. This can result in chaotic nuclear phenotypes affecting their volume. These aberrant nuclear organizations are hampering gall development and nematode maturation. Herein we report on two nuclear volume assessment methods which provide information on the complex changes occurring in nuclei during giant cell development. Although we observed that the data obtained with AMIRA tend to be more detailed than Volumest (Image J), both approaches proved to be highly versatile, allowing to access 3D morphological changes in nuclei of complex tissues and organs. The protocol presented here is based on standard confocal optical sectioning and 3-D image analysis and can be applied to study any volume and shape of cellular organelles in various complex biological specimens. Our results suggest that an increase in giant cell nuclear volume is not solely linked to increasing ploidy levels, but might result from the accumulation of mitotic defects.
Highlights
Plant-parasitic nematodes are a major threat to many plant cultures worldwide, and root-knot nematodes (RKN) are responsible for extensive economic losses caused by phytonematodes (Chitwood, 2003)
Feeding sites induced by RKN are composed of outsized feeding cells named “giant cells” (GCs) which hold a dense cytoplasm filled with organelles and enlarged nuclei, representative of high metabolic activity
These giant-feeding cells are surrounded by mitotically active vascular tissue cells characterized by asymmetric cell wall positioning. This peculiar division pattern gives rise to a multi-layered shell consisting of small cells neighboring the GCs. This outlines the typical knot shape in susceptible roots called a “gall.” On the other hand, Cyst nematodes (CN) initiate a syncytium through elongation of a single initial feeding cell, accompanied by division of neighboring cells which will fuse as syncytia expands (Golinowski et al, 1997)
Summary
Plant-parasitic nematodes are a major threat to many plant cultures worldwide, and root-knot nematodes (RKN) are responsible for extensive economic losses caused by phytonematodes (Chitwood, 2003). Feeding sites induced by RKN are composed of outsized feeding cells named “giant cells” (GCs) which hold a dense cytoplasm filled with organelles and enlarged nuclei, representative of high metabolic activity. These giant-feeding cells are surrounded by mitotically active vascular tissue cells characterized by asymmetric cell wall positioning. Endoreduplication is recognized to affect chromatin dynamics and the number and size of these centromeric heterochromatin domains in interphase nuclei can be indicative of endocycle activity This type of DNA amplification is the major process involved in the increase of ploidy in GCs (de Almeida Engler et al, 1999, 2012; de Almeida Engler and Gheysen, 2013). Comparative volumetric measurements of GC and neighboring cells here named non-giant cells (NGC) nuclei present in wild-type (Col-0) and KRP3OE and KRP5OE Arabidopsis transgenic lines (Coelho et al, 2017) were performed and results are presented
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