Plants interact with a wide range of organisms, often leading to various pathologies. Among these are obligate sedentary endoparasites such as nematodes that establish intimate interactions with a diverse range of plant hosts. Root-knot nematodes (Meloidogyne spp.) are capable of inducing nematode feeding sites within the vascular tissue, containing 5–7 hypertrophied giant cells with multiple abnormally enlarged nuclei. Multinucleate giant cells result from numerous mitotic events in the absence of cytokinesis, and become highly polyploid, possibly by successive endo-reduplication cycles (Sijmons et al., 1994; de Almeida Engler et al., 1999). Cells surrounding young giant cells actively divide to form a multi-layered swelling resulting in a gall. In contrast, cyst nematodes (Heterodera spp.) induce a multinucleate feeding site by stimulating a single vascular cell to become a syncytium through incorporation of adjacent cells via extensive cell-wall dissolution and protoplast fusion (Sijmons et al., 1994; Grundler et al., 1998). Even though galls and syncytia follow a different developmental program, formation of these nematode feeding sites has several features in common. In Arabidopsis thaliana, both types of feeding cells are initiated and localized in the root vascular tissue adjacent to xylem elements. Both contain a dense cytoplasm and numerous large nuclei, undergo organelle proliferation and show elaborate ingrowths of peripheral cell walls (Hussey and Grundler, 1998; Mitchum et al., 2004). Both giant cells and syncytia are polynucleate feeding cells: giant cells via acytokinetic mitoses and syncytia by cell fusion. Due to the presence of enlarged nuclei, it is assumed that additional DNA replication cycles may play a primary role in the establishment of functional feeding cells. Polyploidy in giant cells was first suggested in the 1960s (Owens and Novotny Specht, 1964; Dropkin, 1965), but the mechanism by which it is derived has not been elucidated so far. Previous work has reported that the nuclear DNA content in giant cells increases significantly due to the increased nuclear size and chromosome number (Wiggers et al., 1990; Starr, 1993), possibly leading to drastic feeding cell expansion. Intense DNA synthesis in galls and syncytia also strongly indicated the presence of additional replication cycles in nematode feeding cells (Rubinstein and Owens, 1964; Rohde and McClure, 1975; de Almeida Engler et al., 1999). The endocycle is a variant of the eukaryotic cell cycle in which successive S phases follow each other without intervening mitosis or cell division (De Veylder et al., 2011). The DNA content of the cell is doubled with every new round of DNA replication, resulting in formation of cells with DNA ploidy levels of 4C, 8C, 16C or higher. The endocycle may be induced during biological processes such as cell differentiation, cell expansion, metabolic activity and stress. Plant endoploidy is typically observed in differentiated, outsized cells (e.g. Arabidopsis trichomes), endosperm and fruit (Chevalier, 2007; Larkin et al., 2007; Sabelli et al., 2007).
The plant endocycle is controlled by diverse factors or gene products, and functional analyses of mutants and transgenic plants with aberrant levels of endo-reduplication have led to identification of key regulators of the endocycle. Examples are the two CCS52 classes identified in plants: CCS52A (Cdh1/Fzr/Srw1-type), which is also found in yeast and animals; and CCS52B, which is presumed to be plant-specific (Tarayre et al., 2004). In Arabidopsis, the CCS52A class is represented by two family members (CCS52A1 and CCS52A2), whereas there is only one CCS52B gene (Fülöp et al., 2005). CCS52 is a cell-cycle switch protein that behaves as an adaptor protein that is important for activation of the anaphase-promoting complex/cyclosome (APC/C) and is involved in conversion of mitotic cycles into endocycles. Another example of an endocycle regulator is the plant homologue of the archaeal DNA topoisomerase VI that is required for successful progression of the endo-reduplication cycle in Arabidopsis (Sugimoto-Shirasu et al., 2002, 2005). RHL1 encodes the ROOT HAIRLESS 1 protein and forms a multiprotein complex with plant topoisomerase VI (Sugimoto-Shirasu et al., 2002). Sugimoto-Shirasu et al. (2002) also suggested that topoisomerase VI is required to resolve entangled chromosomes during endocycles above 8C.
In contrast to CCS52 and RHL1, E2Fe/DEL1 (hereafter referred to as DEL1) is an inhibitor of endo-reduplication and preserves the mitotic state of proliferating cells by suppressing transcription of genes required to enter the endocycle (Vlieghe et al., 2005; Lammens et al., 2008). DEL (DP-E2F-like) genes encode atypical E2F-like proteins designated E2F7/E2F8 in mammals. Arabidopsis has three DEL genes (DEL1, DEL2 and DEL3). Loss of DEL1 function results in augmented ploidy levels, while ectopic expression of DEL1 results in decreased endo-reduplication levels.
Plant biotrophic interactors consistently establish specialized interaction sites where nutrient exchange occurs. Augmented plant nuclear DNA ploidy has been described during numerous interactions, including fungal and bacterial symbionts and parasitic fungi and nematodes (Wildermuth, 2010). Here, we address the occurrence of endo-reduplication triggered by two types of nematodes (gall-forming root-knot nematodes and syncytium-forming cyst nematodes) during establishment of their feeding sites, and investigate components potentially participating in this process. The DEL1 and CCS52 gene family and RHL1 may be essential components of the endocycle machinery active in nematode feeding sites in host roots. Therefore, we generated knockdown and over-expression lines of three CCS52 genes and analysed the effect in nematode-infected roots. We also examined comparable lines for DEL1 and RHL1, which have been described as key regulators of the endocycle (Sugimoto-Shirasu et al., 2005; Vlieghe et al., 2005). Data reported suggest that increased DNA ploidy levels in nematode feeding sites are linked to endocycle activation. Collectively, the results support the conclusion that mitotic and DNA replication processes cannot operate independently in favour of a successful nematode feeding site and nematode maturation.