The plant cell inhibitor KRP6 is involved in multinucleation and cytokinesis disruption in giant-feeding cells induced by root-knot nematodes

Abstract

The plant cell cycle inhibitor gene KRP6 has been investigated in roots infected by plant-parasitic root-knot nematodes (Meloidogyne spp.). Unexpectedly, KRP6 overexpressing lines revealed a distinct role for this specific KRP as an activator of the mitotic cell cycle. This function was confirmed in Arabidopsis thaliana suspension cultures ectopically expressing KRP6. A blockage in the mitotic exit was observed in cell suspensions and in giant cells resulted in the appearance of multi-nucleated cells. KRP6 expression during nematode infection and the similarity in phenotypes among KRP6 overexpressing cell cultures and giant-cell morphology strongly suggest that KRP6 is involved in multinucleation and acytokinesis occurring in giant-cells. Once again nematodes have been shown to manipulate the plant cell cycle machinery in order to promote gall establishment.

An insight on KRP cell cycle inhibitors during nematode feeding site development

Root-knot nematodes (RKN), Meloidogyne spp, are distributed worldwide and impose severe economic damage to many agronomically important crops.^1,2 The cell cycle machinery is considered one of the pivotal components for the formation of nematode feeding sites (NFS), that are composed of 5–9 giant-cells surrounded by neighboring cells that are actively dividing.^3,4,5 Proliferating neighboring cells divide asymmetrically around the giant-cells leading to the formation of the typical gall or knot.⁶ These nematode-induced giant-cells undergo synchronous waves of mitotic activity uncoupled from cytokinesis. Successively, the expansion of these giant cells appears to be tightly related to the endocycle, when multiple rounds of DNA synthesis without chromosome condensation or nuclear division occur.^4,7

The orchestration of the host cell cycle machinery by RKN within their feeding sites has drawn particular attention due to the participation of a number of core cell cycle genes.^4,5,8,9 Therefore, studying the cell cycle progression in the NFS will help us to understand the regulatory mechanisms that drive the formation of such specialized feeding sites. Progression through the cell cycle is driven by the cyclin-dependent kinases (CDK) and their regulatory subunits, named “cyclins.”^10,11 The induced transcription of mitotic bona fides CDKA;1 and CDKB1;1, as well as their regulatory cyclins CYCB1;1 and CYCA2;1, illustrated the anticipated stimulation of the cell cycle machinery in nematode-induced galls^4,8, as well as the induced expression of CCS52 gene family members demonstrated their participation into the endocycle occurring in giant cells.^5,12

The plant cell cycle can also be modulated by inhibitors.^11,13,14 In Arabidopsis 7 CDK inhibitors (CKI) belonging to the interactors/inhibitors of CDK (ICK), or also referred as Kip-Related Proteins (KRP) family have been identified.^15,16 Interactions of KRPs and CDK/CYCs complexes decrease CDK activity¹⁷, and affect both cell cycle progression and DNA content in a concentration-dependent manner.¹⁸ Recently, we have investigated the mode of action of different KRPs in galls illustrating that their function may vary among these inhibitor family members.^19,10,21 Deregulation of the cell cycle machinery of NFS via overexpressing or knockout KRP lines were evaluated for a potential cell cycle control during gall development. Our previous data have demonstrated that KRP2, KRP5 and KRP6 are transcriptionally activated in galls, whereas promoter activity of KRP1, KRP3, KRP4 and KRP7 was absent. Ectopic expression of one expressed Arabidopsis KRP gene, namely KRP2, and expression of 2 not expressed KRPs, KRP1 and KRP4, lead to a significant reduction of gall development. Reduced gall size in plants overexpressing KRP1, KRP2 and KRP4 was experimentally linked to the inhibition of both mitotic and endoreduplication activity. This had a direct negative effect on nematode development and offspring.^19,20 Contrary to expectations, our recent data revealed that KRP6 acts as a mitotic activator in plant cells, as well as in galls, when KRP proteins have been essentially identified as cell cycle inhibitors.²²

KRP6 is highly expressed in galls and protein levels fluctuate during NFS development

To obtain further insight into the role of KRP6 in the gall tissues, live-cell imaging and gene functional analysis were combined to investigate how such cell cycle inhibitor could interfere in cell cycle machinery activated in nematode-infected roots. Firstly, promoter-GUS and transcription analysis confirmed KRP6 expression in galls, occurring in both giant-cells and neighboring cells at early stages (1 to 7 d after infection, DAI) of development. At later stages of gall development (>7 DAI) KRP6 promoter-GUS activity was only found coupled to neighboring cells.²² Protein dynamics of KRP6 was followed in nematode-induced galls by confocal microscopy and in vivo observations confirmed green florescent protein-KRP6 (GFP-KRP6) expression in giant-cells. GFP-KRP6 protein fusion accumulation was obvious at early stages of giant-cell formation, associated with the phase of high mitotic activity within giant-cells (Fig. 1A). Absence of GFP fluorescence at later phases of gall development accompanied the increased size of giant-cell nuclei characterizing the endoreduplication phase of giant-cells.

Figure 1.

Functional analyses of the KRP6 Arabidopsis gene in root-knot nematode [Meloidogyne incognita (Kofoid and White, 1919) Chitwood, 1949] induced galls. (A) In vivo localization of GFP-KRP6 in giant-cell nuclei 4 DAI. (B-C) DAPI-stained gall sections at 30 d after infection (DAI) of the KRP6^OE line (B) and wild-type (C). In the KRP6^OE line a significant increase in nuclei number is observed within the giant-cell and ectopic proliferation of neighboring cells is visible. White circles delimit giant cells. Bars: A = 10 μm, B-C = 50 μm.

Enhanced KRP6 levels promote mitotic activity in galls

A second approach that has contributed to our understanding of the role of KRP6 in NFS made use of Arabidopsis cell cultures and stable Arabidopsis plants expressing the 35S:GFP-KRP6 construct. Surprisingly, overexpression of KRP6 in Arabidopsis cultured cells caused the formation of multi-nucleate cells with up to 20 unequally sized nuclei with apparent impairment of cytokinesis.²² Synchronization of KRP6 over-expressing Arabidopsis cultured cells by aphidicolin suggested that ectopic KRP6 expression triggers the competence of an earlier entry into mitosis, however provoking a hindrance in mitosis progression and exit.²² Aphidicolin exerts a blockage of cell cycle progression at early S phase. The observation that constitutive KRP6 expression conduct to a faster mitotic entry of suspension cells, followed by inhibited cytokinesis leading to the formation of multi-nucleate cells, prompted us to further analyze this phenotype in stable Arabidopsis plants. This induced mitotic phenotype was confirmed in Arabidopsis roots stably overexpressing KRP6 upon Meloidogyne incognita (Kofoid and White, 1919) Chitwood, 1949 infection.²² Remarkably, ectopic KRP6 expression resulted in increased nuclei number within giant-cells, as well as augmented proliferation of neighboring cells (Fig. 1B) compared to wild-type (Fig. 1C).

Ectopic KRP6 affects endoreduplication entry in gall tissues and nematode reproduction

Previous analyses of other KRP^OE lines (i.e. KRP1^OE, KRP2^OE and KRP4^OE) showed that the ectopic expression of KRP genes caused severe impairment of mitosis occurring within giant-cells and surrounding neighboring cells, but also prevented proper giant-cell expansion.^19,20 We observed that ectopic KRP6 expression leads to an increase in nuclei number within giant-cells, as well as an increment of neighboring cells number. Galls induced KRP6^OE line were of similar size as in control roots due to ectopic neighboring cell division, however, the corresponding giant-cell surface was significant smaller compared to ones induced in wild-type plants (Fig. 2A). Subsequently we performed flow cytometry analyses to evaluate the ploidy levels in galls induced on KRP6^OE line compared to wild-type (Fig. 2B). As expected, the DNA content of galls induced in the KRP6^OE line (2C to 32C) showed a significant reduction of giant-cell nuclei ploidy levels compared with the wild-type (2C to 64C). This strongly suggested a delay or obstruction of the endoreduplication cycle in giant-cells upon ectopic KRP6 expression. As well, the continuous induction of mitotic events could be associated with a failure of normal progression of the endocycle that most likely promotes proper giant cell maturation. Therefore, changes in ploidy levels during ectopic KRP6 expression indicate that a number of giant-cells were unable to switch properly to the endoreduplication cycle affecting their expansion, and consequently preventing the delivery of sufficient nutrients for suitable nematode development. It is likely that the balance of the mitotic and the endocycle during gall development ultimately control giant-cell maintenance,^3,5 and consequently nematode development and reproduction (Fig. 2C). In galls induced in the KRP6^OE line, the extension of the mitotic phase most likely postponed the start of the endocycle in the reduced-sized giant-cells thus also affecting the nematode reproduction. Once again these data confirm that the multinucleate state in giant-cells is not sufficient to drive giant-cell expansion and completion of nematode's life cycle.^4,5 Therefore, a phase governed by the endocycle seems to be essential for giant-cell expansion.^5,12

Figure 2.

KRP6 overexpression significantly affects giant-cell size and nuclear ploidy levels of galls induced by Meloidogyne incognita (Kofoid and White, 1919) Chitwood, 1949 in Arabidopsis roots. (A) Values are means from measurements of giant-cell surface (μm²) in wild-type plants compared to KRP6 overexpressing line at different stages of nematode infection. Measurements were made on a minimum of 30 giant-cell sections, and 2 to 3 largest giant-cells were randomly measured per gall. Asterisks indicate statistically significant differences at each time point after nematode infection (P < 0.05). Bars represent + 1 SE. (B) Flow cytometry analysis of nuclei extracted from non-infected and nematode-infected roots 30 d after infection (DAI) of wild-type and KRP6^OE plants. (C) Nematode (M. incognita) infection tests showing a significant decrease in egg mass number compared to the wild-type. Asterisks indicate values that were significantly different from the wild type at P < 0.05 (Student's t test).

It is important to point out that 2 loss-of-function KRP6 lines showed an increased frequency of cell wall stubs in giant-cells compared to the wild-type.²² This observation strongly suggests the involvement of KRP6 on the multi-nucleate state of giant cells and on the cell division arrest during the numerous mitotic events occurring in these feeding cells. Mature giant cells in KRP6 knockout lines were also significantly smaller than those in wild-type and contained fewer and more dispersed nuclei, rather than the multiple clustered nuclei seen in normal giant-cells.²⁴ These data together demonstrate the relevance of KRP6 function for giant-cell development.

Concluding remarks

In the past decade significant progress has been made to unravel the molecular mechanisms that lead to the formation of root-knot nematode-induced galls.^23,24,25 The analysis of core components of the cell cycle machinery has shown that the galls form as a result of the competence of nematodes to regulate and usurp this host molecular apparatus. The intricate cell cycle gene modulation by RKN as seen in giant-cells provides an alternative model allowing the study of cell cycle gene function.

In our data generated from nematode induced galls, we have demonstrated a direct link between KRP6 and mitotic activation. Our data have shown an unexpected role for KRP6 during mitosis, demonstrating that different KRPs regulate the plant cell cycle in a distinct way. While KRPs such as KRP1, KRP2 and KRP4 are associated with inhibition of mitosis and thus cell division, we demonstrated that in the case of KRP6 overexpression, the mitotic state of giant-cells is stimulated, leading to their multi-nucleate and acytokinetic state. In addition, irregularly-sized nuclei and nuclear fusion observed in cell cultures ectopically expressing KRP6 might be used to hypothesize the various nuclear sizes in giant cells within their host roots. Cell wall stubs in KRP6 loss-of-function lines, cytokinesis inhibition on cell suspension and root cells of Arabidopsis overexpressing KRP6 also strongly supports its involvement in the cytokinesis inhibition occurring in giant-cells. Altogether, our data support the scene that KRP6 might play a role in regulating giant-cell multinucleation and their acytokinetic state. Since cell cycle components are evolutionary highly conserved, deregulation of certain core components are likely to be exploited by diverse plant pathogens like nematodes to orchestrate host molecular pathways.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We acknowledge all the contributions to this work to Lieven De Veylder and Geert De Jaeger teams, as well as Gilbert Engler.