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

Abstract

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. The establishment and maintenance of physiologically fully functional giant cells are necessary for the survival of these nematodes. As such, giant cells may be useful targets for applying strategies to reduce damage caused by these nematodes, aiming the reduction of their reproduction. We have recently reported the involvement of cell cycle inhibitors of Arabidopsis, named Kip-Related Proteins (KRPs), on nematode feeding site ontogeny. Our results have demonstrated that this family of cell cycle inhibitors can be envisaged to efficiently disrupt giant cell development, based on previous reports which showed that alterations in KRP concentration levels can induce cell cycle transitions. Herein, we demonstrated that by overexpressing KRP genes, giant cells development is severely compromised as well as nematode reproduction. Thus, control of root-knot nematodes by modulating cell cycle-directed pathways through the enhancement of KRP protein levels may serve as an attractive strategy to limit damage caused by these plant parasites.

How do root-knot nematodes alter the host genetic program?

Root-knot nematodes (RKN) are distributed worldwide and have a severe economic impact on many agronomically important crops.¹ Following the induction of the nematode feeding cells by RKN, synchronous waves of mitotic activity uncoupled from cytokinesis, drives the formation of multinucleate giant cells (GCs). Posteriorly, GC expansion seems to be tightly linked to a modified type of cell cycle, known as endoreduplication, in which multiple rounds of DNA synthesis occur without chromosome condensation or nuclear division. This endocycle has been correlated with a consequent hypertrophy of nuclei in giant-feeding cells.^2,3 Adjacent to GCs, the proliferation of neighboring cells will give rise to galls having the typical root-knot phenotype.^4,5

Such host maneuvering leads to permanent changes in the cell physiology suggesting an intricate orchestration of the host cell cycle machinery by RKN.³ A typical cell cycle encompasses 4 sequential ordered phases (G1, S, G2, and M), distinguishing the temporal replication of the cellular genetic material from the segregation of duplicated chromosomes into 2 daughter cells.⁶ Transitions between phases of the cell cycle are controlled by cyclin-dependent kinases (CDKs) acting in complex with their regulatory partners, the cyclins (CYCs), comprising the principal regulators of the cell cycle machinery.⁷ Although the mechanisms of gall formation are highly specific for this family of plant-parasitic nematodes (family Meloidogynidae), studies demonstrate that a similar subset of core cell cycle genes are required as for ordinary root development.^2,3,8,9 Nevertheless, the complex nature and spectrum of nematode targeting host molecular pathways remains to be further elucidated.

Differential involvement of KRP genes in the nematode feeding site development

Plant genomes encode 2 plant-specific families of cyclin kinase inhibitors (CKIs); the ICK/KRPs (interactors/inhibitors of CDK, or also referred as Kip-Related Proteins), and SIM/SMR (SIAMESE) families.^10-13 Interactions of KRPs and CDK/CYCs complexes decrease CDK activity,¹⁴ and affect both cell cycle progression and DNA content, in a concentration-dependent manner.^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 site development (Fig. 1A). The remaining gene members (KRP1, KRP3, KRP4, and KRP7) did not show any significant promoter or transcription activity in gall tissues (Fig. 1B), similarly to what was observed in the root vascular tissue of uninfected roots. Having analyzed promoter activity and transcript localization, protein dynamics of KRP2 was followed in galls by confocal microscopy. These in vivo observations revealed fluctuation of KRP2 protein levels through nematode feeding site development. At early stages of giant cell formation (2–14DAI), a weak fluorescence was observed for GFP-KRP2 (Fig. 1C), associated with the phase of high mitotic activity within the giant cells. At later phases of gall development increased GFP-KRP2 fluorescence in giant cell nuclei was associated with the endoreduplication status of these feeding cells. These fluctuations of protein levels indicate that nematode secretions might control KRP2 protein levels in favor of giant cell development to efficiently nourish the parasitic nematode.

Figure 1. Functional analyses of the Arabidopsis Kip-Related Protein (KRP) family in galls induced by the root-knot nematode Meloidogyne incognita (Kofoid and White, 1919) Chitwood, 1949. (A-B) Differential promoter activity of KRP2 (A) and KRP1 (B) at 14 d after nematode infection (DAI) suggests variable functions of different KRPs on nematode feeding site development. (C) In vivo nuclear (arrows) localization of GFP-KRP2 in giant cell at 14 DAI. (D-F) Overexpression of KRP1 (D) and KRP2 (E) strongly interfere with gall size by impairing the cell cycle. Giant cells are significantly smaller in the KRP1 (D) and KRP2 (E) overexpression lines compared with wild-type giant cells (F), and the decreased mitotic activity drastically decreases neighboring cell division (D-E). Asterisk, giant cell; n, nematode. Scale bars = 50 µm, except C = 5 µm.

We also studied the effect of KRP loss-of-function in single (KRP2^−/−) and double (KRP2^−/− KRP6^-/-) mutant lines, as well as in multi-silenced RNAi lines for 7 KRP genes,¹⁷ during nematode infection. Mutation or attenuation of KRP genes resulted in ectopic activation of mitosis in RKN feeding-sites as illustrated by an increased number of nuclei in giant cells, and abnormal proliferation of vascular cells surrounding these giant cells. This accelerated mitotic activity in galls is consistent with recent data, showing that loss-of-function of multiple KRP mutants promote the accumulation of CDK/CYC complexes, facilitating and triggering plant cell proliferation.^18-20 Increased CDK levels above the normal endogenous cell threshold most likely triggers a shift toward a faster and longer mitotic phase in galls. Despite the accelerated mitotic activity in gall tissues, development of the associated nematodes seems unchanged and these physiological alterations display no synergetic effect to RKN reproduction.¹⁷

Enhanced host KRP levels disrupt cell cycle progression in galls

We are on the course of unravelling components that drive the host cell cycle machinery in GCs induced by RKN. Components involved in DNA replication (G1 and S phases) can be regarded as critical steps for both the mitotic and the endoreduplication cycle in feeding cells. The knowledge that eukaryotic control mechanisms for DNA amplification and nuclear division are conserved in all eukaryotes, prompted us to ask whether accumulation of different KRPs might influence cell cycle progression in developing gall tissues in the host plant. Our rational was to disturb the mitotic and endoreduplication cycle by ectopically expressing cell cycle inhibitors (here KRP genes) to impair gall development, with final aim to control nematode maturation and reproduction. To validate our hypothesis, transgenic Arabidopsis plants overexpressing KRP1 (a non-expressed gene in galls) and KRP2 (a highly expressed gene in galls) driven by the 35S promoter of the cauliflower mosaic virus (CaMV) were generated.

In both, KRP1 (Fig. 1D) and KRP2 (Fig. 1E) overexpressing lines, we observed galls with an overall reduced size and a severe decrease in the number of cells neighboring GCs. Galls lost their typical bulky phenotype obvious in wild-type infected roots (Fig. 1F). This reduced gall size phenotype illustrates a blockage of cell mitotic activity of the proliferative gall cells. Infection studies also revealed a significant decrease in nuclei number in giant cells of both KRP1 and KRP2 overexpressing lines. Likewise, surface measurements illustrate a significant reduction of giant cell volume (Fig. 2).

Figure 2. Inhibition of the cell cycle by overexpression of KRP1 and KRP2 decreases giant cell size. Values are means from measurement of giant cell surface (µm²) in wild-type plants, compared with KRP1 and KRP2 overexpressing lines at different stages of nematode infection. Measurements were made on a minimum of 30 giant cell sections; 2 to 3 largest giant cells were randomly measured per gall. Different letters indicate statistically significant differences at each time point after nematode infection (p < 0.05). Bars represent + 1 SE.

A notable feature of RKN is their competence and resilience to maneuver the host cell cycle machinery in their benefit, as also reported for other mutant lines.^21-23 Even when cell cycle progression was clearly disturbed in gall cells of KRP overexpressing lines, giant cells were still able to develop till a certain size. Nevertheless, galls comprising smaller feeding cells (Fig. 1D and F), and almost devoid of neighboring cells, seems to significantly affect nematode development.¹⁷

Although all KRPs are capable to inhibit CDKA/CYC complexes in plant cells,²⁴ their mode of action can be differentially modulated in galls induced by RKN. 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. These assumptions are supported by the different localization of these proteins in interphase nuclei or along mitosis.^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.^25,26

The ectopic expression of KRP genes (such as KRP2) can have a dual effect on gall development: reduction of mitotic activity and decrease of endoreduplication resulting in reduced gall size. KRP2 is highly expressed in GCs, as well as in the vascular cylinder of roots, where galls are induced. As opposed to KRP2, KRP1 is normally barely expressed in GCs and root tissues although detected in mature leaves containing endoreduplicating cells.²⁷ Although the specificity of the interaction of each KRP with CDKs needs to be further elucidated, published data suggest that CDKA-CYCD complexes, or CDK complexes containing either CDKA or CYCD are the primary targets of KRPs.^28,29 It is conceivable that misexpression of KRP1 could target additional CDK complexes present within gall tissues. Nevertheless, the strongest inhibitory effect observed for KRP2 could be correlated with its preferential expression in root vascular cells. On the other hand, the artificial expression of KRP1 in galls might show its affinity to particular substrates or distinct CDK complexes that are able to deregulate or even block the normal cell cycle in GCs. Local high KRP2 protein levels might facilitate this protein to bind to CDK/CYC complexes inhibiting their activity. Although protein levels in giant cells seem to be controlled during nematode feeding, an increase above endogenous levels of KRP2 in giant cells is sufficient to affect giant cell expansion. Smaller giant cells imply less availability of nutrients, as the majority of nematodes associated with these galls were unable to properly develop and reproduce (Fig. 3).

Figure 3.KRP1 and KRP2 overexpression severely disturb root-knot nematode induced-gall development, and consequently nematode maturation. (A) A typical pear-shaped adult female is normally associated with a bulky gall on wild-type Arabidopsis roots. (B-C) Overexpression of either KRP1 (B) or KRP2 (C) proteins affect cell cycle progression in galls, impairing juvenile root-knot nematode growth and reproduction. Therefore, reduced gall size in KRP1 (B) and KRP2 (C) overexpression lines is evident. G, gall; n, nematode. Scale bars = 50 µm.

Conclusions

Modulating CDK activity is a critical step for both mitotic and endoreduplication cycles.^7,30 Both cell cycle types share common components and cell cycle phases (at least G1 and S) prompting DNA replication. Differences engage mitosis (G2 and M phases) involved in cell division, whereas endoreduplication implies increased nuclear ploidy levels.⁷ It is therefore tempting to target both pathways to modulate progression of the cell cycle in giant cells induced by these highly specialized plant-parasitic nematodes. Cyclin kinase inhibitors, like KRPs, have a dual effect in the mitotic as well as in the endocycle and therefore are interesting candidates to negatively regulate the cell cycle in feeding sites.

Differential promoter activity among the 7 KRPs, differences in giant cell phenotype, and distinct nuclear localization patterns and phenotypes point out that these proteins might differently regulate CDK/CYC complexes. Nevertheless, detailed analysis of the diverse members of the KRP multi-gene family allowed us to select the most effective and robust inhibitor(s) suited for developing new strategies to decrease nematode propagation by inhibiting normal feeding site development. In addition, studying cell cycle gene families in highly specialized cells, like giant cells, showing an amplified and accelerated cell cycle, might help to unravel the specificities of the different cell cycle genes in the plant host. The present findings re-enforce the idea that strategies capable of inhibiting critical core cell cycle components can dramatically affect the structure and size of the gall. We have shown that KRP2 is an important component of cell cycle control in giant cells, and that its overexpression in galls prevents nematode development. Our results demonstrated that increasing KRP levels disarray cell cycle events in galls. These data suggest that core cell cycle components in nematode feeding sites follow a conserved cell cycle track as for the plant host normal development. Therefore, alternative strategies that aim to control nematode damage can be envisaged by using plant cell cycle inhibitors and their regulators.

10.4161/psb.26409