Whole‐mount confocal imaging of nuclei in giant feeding cells induced by root‐knot nematodes in Arabidopsis

Abstract

Summary• Excellent visualization of nuclei was obtained here using a whole-mount procedure adapted to provide high-resolution images of large, irregularly shaped nuclei. The procedure is based on tissue clearing, and fluorescent staining of nuclear DNA with the dye propidium iodide.• The method developed for standard confocal imaging was applied to large multicellular root swellings, named galls, induced in plant hosts by the root-knot nematode Meloidogyne incognita.• Here, we performed a functional analysis, and examined the nuclear structure in giant feeding cells overexpressing the cell cycle inhibitor Kip-related protein 4 (KRP4). Ectopic KRP4 expression in galls led to aberrant nuclear structure, disturbing giant cell expansion and nematode reproduction. In vivo live-cell imaging of GFP-KRP4 demonstrated that this protein co-localizes to chromosomes from prophase to late anaphase during cell cycle progression.• The data presented here suggest the involvement of KRP4 during mitotic progression in plant cells. The detailed results obtained using confocal analysis also demonstrate the potential utility of a rapid, easy-to-use clearing method for the analysis of the nuclei of certain Arabidopsis mutants and other complex plant nuclei.

Función de la Lignina en la Interacción Planta-Nematodos Endoparásitos Sedentarios

“…The mechanisms used to regulate the deposition of new material to the CW and its thickening vary, and as the giant cells form, highly reticulated regions are created from these expansion zones that resemble CW labyrinths called "invaginations", through which the transportation of solutes into and out of the giant cell intensifies (Vieira et al, 2012;Rodiuc et al, 2014). Such invaginations develop throughout the process of maturation of the nematode and degenerate once it reaches its maturity and completes its life cycle (Rodiuc et al, 2014).…”

Enhanced levels of plant cell cycle inhibitors hamper root-knot nematode-induced feeding site development

“…11,15 Recently, we initiated a detailed functional analyses of the Arabidopsis KRP gene family during RKN [Meloidogyne incognita (Kofoid and White, 1919) Chitwood, 1949] infection, in order to reveal their potential involvement on this plant-pathogen interaction. 16,17 Of the 7 Arabidopsis genes, we observed that 3 members of this family (KRP2, KRP5, and KRP6 ) were highly expressed during at least a certain time span of nematode feeding root-knot nematodes (rKn) are highly specialized, obligatory plant parasites. these animals reprogram root cells to form large, multinucleate, and metabolically active feeding cells (giant cells) that provide a continuous nutrient supply during 3-6 weeks of the nematode's life.…”

“…25,26 KRP1 and KRP4 proteins present both nuclear and sub-nuclear localization and co-localize with chromosomes during mitosis. 16,26 KRP2 is evenly distributed in the interphase nucleus and apparently degraded during mitosis. 17 The different sub-nuclear protein localization of KRPs (like KRP1, KRP3, KRP4, and KRP5), suggests diverse roles of these proteins in plant cells.…”

“…Gall tissue undergo intense cell cycle activity, therefore, inhibition of cell cycle progression in the nematode feeding sites seems to have a more drastic effect compared with uninfected plant cells. 2,16,17 Recently, we have shown that overexpression of KRP4 resulted in an aberrant segregation of nuclear DNA, promoting a delay in nematode feeding site development. 16 The variability of nucleus phenotype of the different KRPs challenged with RKN (KRP1, KRP2, and KRP4) may reflect diverse functions or different effects on CDK/CYCs activity.…”

“…2,16,17 Recently, we have shown that overexpression of KRP4 resulted in an aberrant segregation of nuclear DNA, promoting a delay in nematode feeding site development. 16 The variability of nucleus phenotype of the different KRPs challenged with RKN (KRP1, KRP2, and KRP4) may reflect diverse functions or different effects on CDK/CYCs activity. These assumptions are supported by the different localization of these proteins in interphase nuclei or along mitosis.…”