Cajal bodies and their role in plant stress and disease responses

ABSTRACT

Cajal bodies (CBs) are distinct sub-nuclear structures that are present in eukaryotic living cells and are often associated with the nucleolus. CBs play important roles in RNA metabolism and formation of RNPs involved in transcription, splicing, ribosome biogenesis, and telomere maintenance. Besides these primary roles, CBs appear to be involved in additional functions that may not be directly related to RNA metabolism and RNP biogenesis. In this review, we assess possible roles of plant CBs in RNA regulatory pathways such as nonsense-mediated mRNA decay and RNA silencing. We also summarize recent progress and discuss new non-canonical functions of plant CBs in responses to stress and disease. It is hypothesized that CBs can regulate these responses via their interaction with poly(ADP ribose)polymerase (PARP), which is known to play an important role in various physiological processes including responses to biotic and abiotic stresses. It is suggested that CBs and their components modify PARP activities and functions.

Introduction

Cajal bodies (CBs) are highly conserved sub-nuclear structures which are often found physically and functionally associated with the nucleolus (Fig. 1).^1-7 They range in size from 0.2 – 2 µm (depending on the organism) and 1 – 6 of them may be present in a typical nucleus (usually 1-2 in a plant nucleus). CBs are dynamic membrane-less structures which can split into daughter bodies or can fuse together, or localize to nucleoli or move to other parts of the nucleus.^8,9 They have been found to play important roles in RNA metabolism and formation of ribonucleoprotein particles (RNP) involved in transcription, splicing, ribosome biogenesis, and telomere maintenance. In spite of such complexity and multifunctionality, CBs per se are not normally essential for cell survival in either animal or plant organisms.^4,7 However, in some exceptional circumstances such as during embryogenesis in mice and zebrafish, functional CBs are absolutely required for completion of this developmental process and concomitant cell survival.^10,11 It is widely accepted that the reason why cells can generally survive without CBs is probably due to the fact that many CB associated processes can also occur in the nucleoplasm in the absence of CBs.¹² It is thought that CBs can act as docking stations for a plethora of different enzymes and substrates, effectively bringing together diverse cellular components in an organized manner to act as factories for enhancing the speed of individual processes;¹³ a function which may be absolutely essential for embryogenesis in mice and zebrafish, but not for some other activities.

Figure 1.

Confocal image of the N. benthamiana nucleus with CB, as visualized by ectopic Agrobacterium-mediated expression of 2 fluorescently labeled CB markers: U2B″ protein (CB marker) fused to green fluorescent protein (GFP, green) and fibrillarin (a nucleolar and CB marker) fused to red fluorescent protein (mRFP, magenta). The merged image is presented on the right. No, nucleolus (shown by solid line), CB, Cajal body (shown by arrow); the nucleus is shown by dashed line. Scale bars, 5 µm.

It should also be noted that these processes in CBs may be tightly regulated by diverse developmental and environmental cues.^9,14-17 Indeed, different types of stress, such as UV irradiation, heat shock, transcriptional inhibition, osmotic stress, starvation, and viral infection may induce re-arrangements of CBs (such as their number, integrity, structure and architecture).¹⁸ In addition to being responsive to such cues, it has been suggested that CBs could perhaps participate in the transduction of these signals and also those of other biological processes such as cell cycle, development and abiotic stress mitigation,¹⁸ gene silencing and non-sense mediated decay (reviewed in ¹⁹); suggesting new and unexpected functions for these sub-nuclear compartments. Moreover, in just the past decade CBs have been discovered to play a critical role in modulating some virus infections,^20,21 either by providing viruses with some functions required for their replication, or by enhancing host defense mechanisms against viruses. Thus, these enigmatic sub-nuclear bodies are emerging as highly complex and multifunctional regulatory compartments, and model systems including plants, have implicated CBs in a surprisingly diverse range of biological mechanisms which we are only just beginning to understand. Since several excellent reviews have recently been devoted to CB functions in RNA metabolism and RNP biogenesis in both animal^22-25 and plant cells,¹⁹ this review will cover these aspects briefly and will instead focus on non-conventional roles of CBs with particular emphasis on stress responses in plants.

Composition of Cajal bodies

Given their multifunctional character it is not surprising that CBs comprise a broad variety of structurally and functionally diverse protein and RNA species (reviewed in²⁵). In addition to coilin, the most abundant signature protein, the CB proteome includes proteins involved in formation of small nuclear RNPs (snRNP) required for splicing of pre-mRNA [such as survival of motor neuron (SMN) protein, Gemins, Sm], small nucleolar RNPs (snoRNP) involved in maturation (methylation and pseudouridylation) of rRNAs, tRNAs, snRNAs (e.g. fibrillarin) and signaling [small ubiquitin-related modifier (1, SUMO-1); poly(ADP ribose)polymerase (PARP) and poly (ADP-ribose) glycohydrolase (PARG)]. CBs in plants also contain components involved in RNA silencing (Dicer 3-like, AGO 4, Pol V, RDR2). Many of these proteins are known to interact with themselves or with other CB components, thus creating a potentially dense network of interactions, which may hold the entire structure together. CB proteins found so far in plants are listed in Table 1.

Table 1.

Protein components of plant Cajal bodies.

Role/Function	Protein	References
Major scaffolding protein of CBs	Coilin	4
SnRNP (maturation and splicing)	U2B″ Spliceosomal protein, component of U2snRNP	3
	SMN Survival motor neuron protein: plant homolog	108
	Gemin 2	109
	Sm	110
	PHAX Phosphorylated adaptor for RNA export	111
SnoRNP (maturation)	Fibrillarin 2′-O-ribose methyltransferase	83,112
	Dyskerin Pseudouridine synthase	113
Gene silencing	Dicer-like 3 RNaseIII-like enzyme	68
	AGO 4 ARGONAUTE4	65
	Pol V DNA-dependent RNA polymerase V	68
	RDR2 RNA-dependent RNA polymerase 2	68
Signaling/stress responses	PARP Poly(ADP-ribose) polymerase	S. Makarova, N. Petukhova and N. Kalinina, unpublished results

Among RNA species found in CBs are various small non-coding RNAs such as snRNAs, snoRNAs and small Cajal body-specific RNAs (scaRNAs; which have functions similar to snoRNAs in RNA maturation). Remarkably, CBs in plant cells may also comprise poly(A) RNAs, including mRNAs.²⁶ Functional activities of plant CB components are discussed in sections below.

Coilin and CB assembly

The major scaffold protein component of CBs is coilin, which is essential for CB formation and function. It has previously been demonstrated that knockout, knockdown or mutation of coilin homologues in Drosophila,⁷ mice,^2,10 zebrafish,¹¹ Arabidopsis⁴ or Nicotiana spp²¹ can prevent CB formation. Analysis of coilin from these different organisms has indicated that there is low amino acid homology (in particular in the middle region; the N- and C-termini are more conserved; see Fig. S1), but there is similar domain organization (Fig. 2). For example, coilin from different species typically contains 2 nuclear localization signals and a putative nucleolar localization signal in the central part of the protein.^8,27-30 Other common domains among the different coilins are an N-terminal globular domain, a C-terminal putative Tudor-like structure abutting a disordered C-terminus, and a highly disordered central domain.³⁰ Analysis of an Arabidopsis thaliana coilin found that it was able to bind RNA strongly and non-specifically; activities ascribed by the 2 sets of basic amino acids in the N-terminal region and a set in the disordered central region.³⁰ These data are consistent with other studies, which show that mammalian coilin interacts with RNA in vivo, effectively binding hundreds of small non-coding RNAs (snRNAs, snoRNAs and scaRNAs).³¹ This suggests that coilin has a broadly conserved dynamic regulatory function, which permits CBs to act as a cellular center of small RNA metabolism.

Figure 2.

Schematic representation of the functional domains of coilin from Arabidopsis thaliana. NOD, an N-terminal ordered domain which is suggested to be responsible for self-association. IDD, an internal disordered domain encompassing 2 nuclear localization signals (NLS1 and NLS2). CTD, a C-terminal domain which contains a presumable Tudor-like structure. Amino acid positions are indicated below.

Upon binding RNA it was found that the N-terminal globular domain of Atcoilin underwent a conformational change which catalyzed multimerization into aggregates. This is consistent with other reports which have found that the N-terminal 92 amino acid sequence of coilin is critical for CB assembly, self-interaction, targeting to CBs, and de novo CB formation.^27,32,33 It is thought that this process is also influenced by 10 residues of the most distal end of the C-terminus, which may control N-terminus self-interaction, which may in turn regulate coilin localization,³⁴ CB formation and number.³⁵

Possible mechanisms of coilin involvement in CB assembly have recently been discussed in detail by Machyna et al.^25,36 Briefly, the model suggests that CBs may be nucleated by tethering different CB components, proteins or RNAs, to a specific site (usually transcriptionally active region) inside the nucleus in a stochastic manner.^37-39 Presumably multiple types of RNA (such as snRNAs, snoRNAs, and/or scaRNAs) arising from separate genomic loci may act as distinct “seeds” for further nucleation of CBs via association of specific sets of proteins for particular RNA species. Thus a group of pre-CB components would be formed as independent entities^22,25 and later on assembled into mature CBs by a bridging protein, most probably coilin. Indeed, coilin can directly interact with many CB proteins and RNAs, thereby acting as a molecular “glue” which brings numerous distinct pre-CB components together via numerous protein-protein and/or protein-RNA interactions.^22,25 It is known that the symmetrical dimethylation of several arginine residues and the phosphorylation state of coilin can control its capacity to bind other CB proteins and RNAs, and this in turn is thought to influence pre-CB seed coalescence and maintenance.^40,41 This “multiseeding” model fits well with the observation that upon coilin knockout or depletion, cells will lack canonical CBs but instead contain several types of residual bodies.²⁵

There are some other sub-nuclear bodies which resemble CBs by shape, size, intra-nuclear location, and also share some protein and RNA components. One of the key differences between these bodies and CBs is dependence of assembly on coilin. An example is the Histone locus body (HLB) which is known to be involved in processing of histone pre-mRNAs.⁴² Coilin is present in a substantial portion of HLBs (though not in all), but in contrast to CBs, HLBs are unaffected by coilin depletion.⁷ Some other bodies may contain no coilin at all, but share other CB components; for example in plants Dicing bodies (or D-bodies) totally lack coilin, but contain other CB residents (e.g., SmD3/SmB). These bodies also comprise the nuclear RNA-binding protein HYL1 and RNase III enzyme DCL1 (Dicer-like 1) with important roles in production of miRNAs, which can in turn regulate gene expression via gene silencing mechanisms.^43-45 It is not clear why different sub-nuclear bodies contain the same components; they just may be in different functional complexes, or shuttle between these compartments. Thus, defining nuclear bodies based on the presence of certain constituents seems to be difficult, since many of them can be present in other types of organelles as well. However, most researchers in the field would agree that CBs are sub-nuclear compartments containing coilin, and coilin is absolutely required for CB assembly and integrity.

Apart from its structural role in the formation of CBs, new important functions of coilin have recently been discovered and are discussed in the sections below. Functional activities of other proteins listed in Table 1 are also reviewed below.

CB functions in RNA metabolism and RNP biogenesis

snRNPs. One major process that is intrinsically linked to CBs is formation of spliceosomal particles, snRNPs, which control pre-mRNA splicing. Spliceosome formation begins with synthesis and migration of major U snRNAs (such as U1, U2, U4 and U5) to CBs for recruitment of the export factors (PHAX and CRM1), which then transport snRNAs to the cytoplasm.⁴⁶ In the cytosol, snRNAs associate with Sm proteins arranged into heptameric rings, and undergo hypermethylation of their 5′ caps. Specificity of the interaction between snRNAs and Sm proteins is controlled by the SMN complex composed of the survival motor neuron protein (SMN) and Gemin proteins. The Sm and hypermethylation modifications target the complex back into the nucleus via a snRNP import receptor. An additional snRNA, U6, is also produced however it does not migrate to the cytosol, but instead it is retained in the nucleus where it is modified by addition of a monomethyl cap. It was reported by Beven et al.³ that in the nuclei of Pisum sativum these macromolecular complexes became associated with CBs, which is consistent with other later findings which additionally suggest that this association mediates maturation of snRNPs prior to their participation in pre-mRNA splicing in the nucleoplasm.¹⁹ In this complex, scaRNAs (described in more detail below) which contain CB localization signals⁴⁷ guide maturation by promoting the pseudouridylation and methylation of snRNAs.^48,49

snoRNAs and scaRNAs

Another major class of small RNAs found in CBs are snoRNAs and scaRNAs that are related in structure and primarily guide chemical modifications of other RNAs, mainly rRNAs, tRNAs and snRNAs (reviewed in^19,25). There are 2 main types of snoRNA, the C/D box snoRNAs and the H/ACA box snoRNAs which have different functions. The C/D box snoRNAs are associated with fibrillarin (methyltransferase) and other additional proteins to form snoRNPs which direct 2′-O- methylation of RNA targets. The H/ACA box snoRNAs forms a complex with dyskerin (pseudouridine synthase) which guides pseudouridylation of specific nucleotides. scaRNAs are abundant in CBs, contain either or both of the boxes together and modify spliceosomal snRNAs during their final maturation inside the CBs (see above). In plants several putative scaRNAs were identified through library screening⁵⁰ and these were later cloned and found to localize specifically to CBs,⁵¹ indicating common modification mechanisms among different species. These modifications are thought to be typically confined to regions of the snRNA important for interaction with pre-mRNAs, other snRNAs or spliceosomal proteins.⁵² Such interactions with diverse components culminate in the production of fully functional U2, U4/U6 and U4/U6-U5 snRNPs, which are finally liberated from the CB for splicing in the nucleoplasm. Some of these complexes are dissociated during the splicing process and these maybe retargeted to the CB for regeneration.

Since both sno- and scaRNAs and their associated proteins co-localize inside CBs, it was proposed that CBs might play a role in maturation of this type of RNPs.

Telomerase

A telomere is a section of DNA at each end of a chromosome that contains repetitive nucleotide sequences which stabilizes and protects the end of the chromosome from fusion with other chromosomes and inappropriate attack by DNA modifying machinery. In plants and other organisms the telomere length is preserved predominantly via the activity of telomerase, an RNP-based enzyme which consists of telomerase RNA (TR), telomerase reverse transcriptase and other associated proteins (reviewed in^19,25). It is thought that loss of activity of this enzyme may constitute a mechanism of aging. In most organisms the catalytic subunit of this complex (TERT) is encoded by one gene, but in plants it is possible that multiple TERT variants are expressed, as indicated by studies on the allotetraploid Nicotiana tabacum (tobacco), where 3 gene variants are present (reviewed in⁵³). Plant TERTs possess multiple nuclear localization/export signals which may explain their localization in the nucleus and the nucleolus.⁵³ It would be expected that in these locations the TERTs would interact with various protein partners to facilitate modulation of telomere length, however only very few interactors (such as plant telomeric dsDNA binding proteins and plant homologs of human telomeric ssDNA binding protein) have been elucidated in planta; this is in contrast to yeast and mammalian interactome studies in which an abundance of TERT interactors have been uncovered.⁵³ Interestingly, in invertebrates the TR which co-purifies and is closely associated with the TERT, contains a specific CB-targeting CAB box. In human cells, CB localization of TR is absolutely essential for telomere maintenance (reviewed in²⁵). While the CAB box has not been reported in plant TRs, it has been shown that Arabidopsis TR is able to interact with dyskerin which is known to be a component of CBs. Thus, the challenge would be to elucidate if the plant telomerase pathway includes CBs.

Although the maintenance of telomeres is predominantly carried out by telomerase (an RNP-based enzyme responsible for telomere length), in some organisms such as plants, there are additional mechanisms such as telomerase independent alternative telomere lengthening (ALT) and recombination dependent machinery which can participate in this role.⁵³ With some organisms such as Arabidopsis, it was indicated that if there is a deficit in telomerase, the other systems such as the ALT pathway can compensate and elongate the telomeres. This study also suggested that ALT mechanisms may play a role in early plant development.⁵³ However, the role of the telomerase independent pathways are poorly studied from the context of CBs/coilin in plants.

New plant-specific CB functions in RNA regulatory pathways

Nonsense-mediated mRNA decay (NMD)

The NMD pathway is a quality control mechanism that recognizes and degrades aberrant (truncated) mRNAs with a premature termination codon. Several lines of evidence demonstrate that in plants NMD may occur in the nucleolus whereas in animals this process is associated with cytoplasmic processing bodies.⁵⁴ Firstly, it has been shown that Arabidopsis exon junction complex proteins accumulate in the nucleolus,⁵⁵ suggesting a role for the nucleolus in mRNA production. Secondly, the plant nucleolus has been reported to contain mRNAs, including properly spliced, aberrantly spliced, and single exon gene transcripts. Aberrant mRNAs are significantly more abundant in the nucleolus, while fully spliced products are more abundant in the nucleoplasm.⁵⁴ The majority of the aberrant transcripts contain premature termination codons and exhibit characteristics of NMD substrates.⁵⁴ In addition, the NMD Up-frameshift (UPF) factors, UPF2 and UPF3 are co-localized with a nucleolar marker protein, fibrillarin, in the nucleolus, suggesting that the Arabidopsis nucleolus may be involved in recognizing aberrant mRNAs and NMD.⁵⁴ Finally, direct correlation between the accumulation of increased levels of aberrant mRNAs in the nucleolus and their turnover by NMD has been demonstrated using upf mutants, which have been shown to be impaired in NMD such that mRNAs that are normally turned over by NMD accumulate in these mutants.^56,57 Given that fibrillarin is a component of CBs and CBs are often physically and functionally associated with the nucleolus, it has been suggested that CBs might also be involved in some aspects of NMD.¹⁹ This suggestion is supported by recent observations showing that CBs contain poly(A) RNAs including mRNAs.²⁶ However, further research is required to explore if direct functional links between the nucleolus and CBs really exist to provide a fully operational NMD pathway.

Gene silencing

Several recent reports implicate CBs as also being involved in modulation of plant gene expression via the biogenesis of microRNA (miRNA) and small interfering RNA (siRNA). These RNA species can trigger transcriptional and post transcriptional gene silencing (TGS and PTGS, respectively) to control gene expression by affecting mRNA production or degradation (reviewed in^58,59). The mechanism of TGS and PTGS is typically induced in response to the presence of aberrant mRNA structures (such as double stranded RNA or RNA with hairpin loops for example) which may arise from endogenous genes, virus infection or introduced transgenes. When aberrant RNAs are present they are typically converted into dsRNA (if not already) by eukaryotic or viral RNA-dependent RNA polymerases (RDRPs),^58,59 prior to their cleavage into 21-24 nucleotide (nt) dsRNAs by distinct Dicer or Dicer-like (for plants) enzymes.^58,59 For PTGS, short 21/22 nt dsRNA fragments become single stranded (mature siRNA and miRNA) and associate with ARGONAUTE proteins (AGO) to form the RNA-induced silencing complex (RISC), which targets mRNA species containing complementary sequences for degradation, impinging on the expression of these particular genes.^58,59 In the case of siRNA, one specific mRNA target is degraded, whereas complexes consisting of miRNA can have multiple targets which can affect mRNA species of different genes. With regard to TGS, it has been suggested that both 21 and 24 nt dsRNAs can associate with AGO proteins to target chromatin-associated scaffold transcripts in a sequence specific manner.^60-62 The complexes which become attached to chromatin subsequently recruit DRM proteins to methylate the DNA at cytosine in CHH, CHG and CG sequence contexts.^63,64 These methylation patterns interfere with transcription of the target gene through various mechanisms.⁶⁵

In Arabidopsis thaliana it has been shown that components of the silencing machinery, such as AGO4 and Dicer-like 3 can co-localize with CBs in the nucleus.^43,66,67 Moreover it has been demonstrated that the major component of RDRP Pol V, which transcribes noncoding and intergenic sequences to modulate heterochromatin formation and silencing of overlapping and adjacent genes,⁶⁸ co-localizes with AGO4 to CBs in Arabidopsis. RDRP2 which is essential for synthesis of siRNAs from retro-elements and transposons, and short distance cell-cell spread of the transgene silencing signal, has also been found associated with CBs in plants.⁶⁹ Taken together this body of data is suggestive that CBs may operate as processing centers in plants which generate RNA species involved in silencing.¹⁹ In addition, it has been found that in an Arabidopsis coilin mutant which has impaired production of CBs, there is significant reduction of overall AGO4 protein levels, suggesting that CBs (or coilin itself) may play a role in stabilizing AGO4 protein.⁶⁷ Finally, coilin and dicer-like 3 double mutants have exhibited a small decrease in DNA methylation beyond that seen in dicer-like 3 single mutants, but has not decreased methylation to the levels detected in ago 4.⁶⁷ These data suggest that although CBs and/or coilin itself, may be required for a fully functioning DNA methylation system which controls TGS in Arabidopsis, the function of CBs may still partially remain in the coilin mutant. It is possible that if formation of CBs is disrupted, their function in DNA methylation may still be fulfilled by other sub-nuclear bodies, e.g. pre-CB structures which may be formed by CB components even in the absence of coilin (coilin is required only for gluing these structures into CBs, as discussed above).

Newly identified functions of plant CBs in responses to stress and disease

Cells rapidly respond to stress in a variety of ways by altering their metabolism either to activate survival pathways or to initiate cell death or apoptotic mechanisms that eventually eliminates damaged cells. Stress also often results in inhibition of major nuclear processes (e.g., DNA replication and transcription) and reorganization of nuclear structure and architecture.¹⁸ The role of CBs in stress-sensing mechanisms in mammalian cells has been discussed in detail by Boulon et al.¹⁸ and will therefore be complemented here by recent advances in studies of plant CBs in the context of biotic and abiotic stresses.

Virus infections

Viruses can enter plants either through mechanical damage, soil microbes or via feeding by nematode and insect vectors. Following entry into cells, replication occurs, which precedes local cell-to-cell and long distance movement of the virus through the plant. Such movement requires virions or nucleic acid/protein complexes to translocate into neighboring cells via plasmodesmata, which are intercellular conduits which links the cytoplasm of contiguous cells.⁷⁰ Through local spread the virus eventually reaches phloem vessels, a vascular network which transports photoassimilates and macromolecules throughout the plant, facilitating systemic dissemination of the virus through the plant.^70,71 For viruses to actively invade plants they have to subvert existing plant cell processes and machinery for replication and dissemination, while counteracting anti-pathogen defense systems.

The nucleolus plays a crucial role in the infection cycle of numerous DNA and RNA-containing viruses (reviewed in^72-74). Since DNA viruses tend to replicate within the nucleus it is easier to comprehend their associations with the nucleolus. RNA viruses replicate mainly within the cytoplasm of host cells so it is unexpected for them to be targeted to this organelle. However, the interactions with the nucleolus are thought to be a universal “pan-virus” phenomenon (reviewed in^72-74), such that plant viruses do not differ from other eukaryotic viruses in this regard.⁷⁴ Much less is known about virus interactions with CBs.

One plant virus that targets the nucleolus is groundnut rosette virus [GRV, a single-stranded (ss) RNA virus belonging to the family Umbravirus]. In contrast to most plant viruses, umbraviruses do not form conventional virus particles as they do not encode a coat protein (CP) which is often required by viruses to enable them to move systemically throughout their host. However instead, GRV encodes the ORF3 protein which compensates for the lack of conventional CP.^75-78 This protein shows no similarity to any viral or non-viral proteins (with the exception of the corresponding proteins of other umbraviruses) and acts as a long-distance movement protein which can bind viral RNA to form filamentous RNP particles that protect the RNA from degradation.^76,77 These particles accumulate as inclusions in the cytosol and are also thought to shuttle through the phloem to distribute the viral genome throughout the plant.^76,78 In addition to the ORF3 protein localizing to cytoplasmic inclusions, it has also appeared to interact with nuclei and is preferentially targeted to nucleoli.^75,79

ORF3 localization to the nucleolus is thought to occur via interaction with reorganized multiple CB-like structures (CBLs) which merge with the nucleolus (Fig. 3).⁸⁰ The mechanisms by which ORF3 targets to CBs and promotes the formation of CBLs remain unelucidated, but likely involve ORF3 hijacking and subverting existing CB transport and distribution pathways to control CB form and function. For example, ORF3 may induce the redistribution of CB components (such as coilin, fibrillarin and U2B”)⁴ by modulating the phosphorylation of coilin^81,82 or the self-association and interaction of CB and other nucleolar components.^16,83 ORF3 eventually migrates from the nucleus to the cytoplasm, and during this process it sequesters fibrillarin and deposits it in the cytosol, a location where it is not normally found.^80,84 It was hypothesized that fibrillarin may contribute to the formation of cytoplasmic filamentous ORF3 RNPs and the concomitant long-distance systemic movement of these viral RNP complexes. Fibrillarin knockdown experiments showed that fibrillarin is required for both these processes, but not for the replication or local cell-to-cell spread of the virus.⁸⁰ In vitro work found that ORF3 can assemble into structures similar to the filamentous viral RNPs formed in planta, when mixed with fibrillarin and viral RNA; these structures were also shown to be infectious.^80,84 Formation of these assemblies requires the interaction of the ORF3 leucine-rich region with the fibrillarin GAR domain.^80,85 It was later revealed from atomic force microscopy studies that the fibrillarin-ORF3 complexes form single layer rings of 18-22 nm which encapsidate viral RNA to form a helical morphology.⁸⁶ These structures, when formed in phloem companion cells, can enter sieve elements and move through the plant to cause systemic infection. Thus, umbraviruses can hijack host rRNA and RNP processing machinery to perform unexpected functions in unusual cellular locations to facilitate long distance virus spread. However, the molecular mechanisms of these processes remain unknown. One possibility involves the fact that fibrillarin can be post-translationally modified by asymmetric arginine demethylation (reviewed in²⁴). Arginine methylation often affects protein-protein interactions and impacts a variety of cellular processes, such as protein trafficking and signal transduction⁸⁷. Further research is necessary to elucidate if such modification is associated with fibrillarin function in umbravirus movement.

Figure 3.

Model of GRV infection. Upon GRV infection, the ORF3 protein enters the nucleus and is targeted to CBs, reorganizing them into multiple CB-like structures (CBLs). The CBLs then move to and merge with the nucleolus via an unknown mechanism. Some host proteins are probably involved in targeting the ORF3 protein to the CBs, reorganizing CBs and causing their fusion with the nucleolus. One such host protein is fibrillarin, which is eventually partially relocalized into the cytosol by the subsequent migration of the ORF3 protein to this destination. In the cytoplasm viral RNP complexes containing the ORF3 protein, fibrillarin and viral RNA form. When produced in the companion cells of the phloem (a plant specific transport system used for trafficking assimilates and macromolecules), the viral RNPs migrate into the phloem sieve elements (conducting cells) where they are transported to the rest of the plant to generate a systemic infection.

In order to further understand the role of CBs in plant viral infection pathways, Shaw et al.²¹ knocked down (K_D) coilin in Nicotiana plants and found that this could differentially modulate plant responses to a diverse range of virus taxa. For example, it was shown that coilin K_D/CB depletion increases accumulation of barley stripe mosaic virus (BSMV; ssRNA rod shaped virus, hordeivirus) and tomato golden mosaic virus (TGMV; DNA spherical virus, begomovirus) which may concomitantly enhance systemic virus spread. Interestingly, coilin/CB deficiencies were also shown to impinge on recovery of newly emergent leaves from symptoms induced by tomato black ring virus (TBRV; ssRNA spherical virus, nepovirus) and tobacco rattle virus (TRV; ssRNA rod shaped virus, tobravirus). The recovery phenomenon occurs due to induction of silencing mechanisms which attack viral genomes in new tissues; it was thus intriguing that depletion of coilin/CBs could influence this process in the case of TBRV and TRV. However, in contrast to expectations, the levels of TRV and BSMV specific siRNAs (the main hallmark of RNA silencing) have not been shown to be reduced in coilin K_D plants. This indicates that silencing initiation is not inhibited in coilin deficient plants and that they are able to trigger specific silencing against TRV or BSMV at levels comparable with WT plants. Therefore it has been suggested that coilin/CBs may be involved in an unknown host defense mechanism operating in addition to RNA silencing.²¹

While it seems that coilin/CB reduction may mediate plant defense responses to enhance infections by different viruses species, the same study²¹ also reported the opposite phenomenon, where coilin/CB K_D were found to reduce symptom development and accumulation of turnip vein clearing virus (TVCV; ssRNA rod-shaped virus, tobamovirus; Fig. 4) and potato virus Y (PVY; ssRNA filamentous virus, potyvirus). Collectively these data suggest that coilin/CBs are important in regulating virus pathogenesis in plants. Although the underlying mechanisms remain to be elucidated, these observations suggest that there might be interplay between coilin (and CB) activities and plant antivirus defense. Disease outcome in virus infection is determined by the race between host defense and virus replication and spread. Functions of coilin and /or CBs can presumably contribute to either side in this race, implying that the mechanisms involved are complex and interwoven.²¹ A possible activity of coilin may be to directly interact with viral proteins, as was shown for virus proteins encoded by poa semilatent virus, which is closely related to BSMV.⁸⁸ Collectively these data suggest that coilin (CBs) may have new functions that are either exploited by plant viruses for their own benefit or are involved in antivirus plant defense responses. These functions may also be involved in other biological processes, such as stress responses.

Figure 4.

Example of symptoms induced by turnip vein-clearing virus in WT (left panel) and coilin K_D (right panel) N. benthamiana plants. Images are courtesy of Dr J. Shaw (JHI).

Abiotic stress

In mammalian cells, various types of both biotic and abiotic stress often affect the nucleolus by inducing a complex range of changes in its morphology, size and protein content.^18,72 Moreover, proteomic studies have uncovered a whole network comprising various nucleolar proteins involved in stress responses.^18,89-91 Altogether, these findings suggest that the mammalian cell nucleolus can operate as a stress sensor and major hub for coordinating stress responses.^18,20 Given that CBs are physically and functionally associated with the nucleolus, it is seems conceivable that CBs in concert with the nucleolus, may play a role in stress sensing and signaling pathways (reviewed in¹⁸). Indeed, different types of stress such as UV irradiation, osmotic stress, transcriptional inhibition, heat shock and starvation, alter structure of CBs and redistribute coilin and other CB components.¹⁸

It has previously been reported that plant cells exposed to high salt (NaCl), can induce osmotic shock, imbalance in the cellular ion concentration and oxidative stress,⁹² which are manifested by increased H₂O₂ production, accumulation of programmed cell death (PCD) markers, cytosolic cytochrome c leakage and bleaching.^93,94 In order to determine whether coilin and/or CBs can influence such responses to abiotic stress, coilin silenced K_D transgenic or non-transgenic tobacco leaf discs were treated with increased concentrations of NaCl. In the non-transgenic discs, application of salt resulted in the loss of their viability and pigmentation (bleaching) (Fig. 5A; S. Makarova, N. Petukhova and N. Kalinina, unpublished results). In contrast, with the coilin K_D transgenic leaf discs, enhanced viability (suppressed bleaching) of leaf discs and impaired H₂O₂ accumulation was observed (Fig. 5A; S. Makarova, N. Petukhova and N. Kalinina, unpublished results). The toxic effect of salt stress at the whole-plant level was also considerably attenuated in coilin K_D plants (Fig. 5B; S. Makarova, N. Petukhova and N. Kalinina, unpublished results). Taken together this data could indicate that suppression of coilin gene expression can confer salt tolerance. Stress signaling pathways in plants comprise complex processes involving various mechanisms, and CBs (or coilin) appear(s) to be a previously unrecognized major player in these pathways. Thus, besides their primary role in RNA metabolism and RNP biogenesis, CBs may have additional functions in plant perception and responses to stress.

Figure 5.

Effect of coilin deficiency on salt stress responses in transgenic N. benthamiana plants. (A) Leaf disks were incubated in salt solution (250 mM NaCl) or water over the course of 10 d at room temperature. K_D, coilin knock down; WT, wild type. (B) Whole WT (left) and coilin K_D (right) plants were watered with 300 mM NaCl solution over 10 d. Images are courtesy of Dr S.S. Makarova (Lomonosov Moscow State University).

Role of poly(ADP-ribose) polymerase (PARP)

New insights into molecular and cellular functions of CBs in plant biotic (e.g., virus attack) and abiotic stress responses may come from recent studies showing a close association of CBs and the PARP family member, PARP1, in Drosophila cells.⁹⁵ PARP1 is a nuclear protein which plays important roles in genotoxic stress tolerance and DNA repair, transcription, cell cycle control and cellular responses to biotic and abiotic stresses including PCD-related processes.^19,95-99 All these regulatory functions can be achieved via the basic enzymatic activity of PARP1, which is involved in post-translational modification of specific proteins. PARP1 catalyzes the attachment of multiple chains of ADP ribose [poly (ADP) ribose; PAR] from NAD to target (acceptor) proteins, and the main acceptor is PARP1 protein itself.^96,97 Interestingly, most of the PARP1 molecules bind to chromatin and accumulate in the nucleolus. However, upon automodification with PAR, PARP1 interacts with key components of CBs, such as coilin and fibrillarin.⁹⁵ This association mediates the shuttling of the PARP1 and other PARylated target proteins from chromatin and nucleolus into CBs for recycling by PAR glycohydrolase (PARG) which hydrolyzes PARylated proteins to free PAR or mono(ADP-ribose).⁹⁵

Plant and mammalian PARPs have essentially similar structures suggesting that their enzymatic activities are conserved across the plant and animal kingdoms.⁹⁷ In plants, these activities have now been implicated in several physiological processes, including responses to abiotic and biotic stresses.⁹⁷ For example, using RNAi approach it has been shown that PARP suppression in Arabidopsis and Brassica napus (oilseed rape) plants results in enhanced stress tolerance to drought, high light and heat stress.¹⁰⁰ Overexpressing PARP in soybean cells is protective against low ROS concentrations, but exacerbated cell death at high ROS concentrations.¹⁰¹ In addition, PARP inhibitors protect soybean and tobacco cells from oxidative stress and heat shock induced PCD.^101,102

Poly(ADP-ribosyl)ation also has substantial impacts on plant-pathogen interactions.

In particular, PARP knockout mutants of Arabidopsis have been shown to exhibit enhanced susceptibility Pseudomonas syringae pv. tomato (Pst) strain DC3000¹⁰³ suggesting that PARP is required for antibacterial resistance. Consistent with this finding, PARP inhibitors have been reported to block certain plant basal defense mechanisms including cell wall reinforcement with callose and lignin, which are induced by microbe-associated molecular patterns, such as bacterial flagellin or EF-Tu epitopes.¹⁰⁴ Both callose deposition and fortification of cell walls with lignin may be important factors in conferring resistance against various plant pathogens.⁹⁷ For example, callose deposition may impose a direct physical barrier that restricts intercellular trafficking of the virus through the plasmodesmata.¹⁰⁵

Plant PARPs have also been implicated in differentiation and cell cycle control pathways,⁹⁷ which are known to overlap with components of plant signaling in response to stresses. Alterations in the poly(ADP-ribosyl)ation level induced by extrinsic (biotic or environmental) or intrinsic (genetic/physiological) cues play a central role in PARP-mediated cellular stress and developmental signaling processes;^96,97 however the detailed molecular mechanisms underlying the these processes remain largely uncharacterised. Given that the interaction of PARP with coilin and fibrillarin seems to be a key factor in trafficking the automodified PARP and other PARylated proteins to CBs for recycling,⁹⁵ it appears reasonable to hypothesize that redistribution of coilin and fibrillarin as well as changes in CB structure, number and content, induced by biotic/abiotic stress or developmental factors, may directly modulate accumulation and localization of PARP, target PARylated proteins and PAR. It seems further conceivable that these factors trigger and propagate multiple signals controlling various stress responses and developmental processes. The corollary is that CBs (and their components) may act as a stress sensor which can modify PARP activity in order to activate various responses to biotic and environmental stresses and developmental cues. It is thus intriguing to speculate whether the coilin-mediated plant anti-virus defense mechanisms described above are instigated by modified PARP function, and also to what extent CB-PARP interactions could control plant responses to other biotic and abiotic stresses; potential pathways which warrant future investigation.

Conclusions

Plant CBs like their animal counterparts, are distinct sub-nuclear bodies involved in coordinating major processes of RNA modification and RNP biogenesis. While the role and mechanisms of CBs in influencing these processes have been well elucidated, recent findings have indicated that CBs have additional new cellular functions which remain largely uncharacterized.

In particular, based on the functional and physical association between the nucleolus and CBs it has been suggested that plant CBs may take part in nonsense-mediated mRNA decay in concert with the nucleolus. However, further research is required to assess if NMD actually occurs in CBs and which precise functions are associated with these sub-nuclear bodies.

Another RNA regulatory process which is often attributed to plant CBs is gene silencing. Gene silencing refers to several mechanistically related transcriptional and post-transcriptional pathways which are involved in controlling and regulating gene expression. It is becoming obvious that not all of them are affiliated with CBs. In particular, our preliminary results have not found any direct links between virus induced silencing and CB functions.²¹ An outstanding question is therefore which silencing pathways are regulated by CBs, and how CBs integrate these processes?

The results of the involvement of plant CBs in regulation of plant responses to biotic and abiotic stresses are especially intriguing, since they have provided a new tentative concept hypothesizing that CBs may sense these stresses and activate PARP-related processes to control them. There is remarkable inter-kingdom functional conservation of PARP and CBs. Both PARP and CBs have been implicated in responses to a wide range of biotic and abiotic stresses. Furthermore, PARP is known to be an important factor involved in the development of defense responses to some human and animal viruses.^106,107 Whether CBs operate as a mediator for sensing and triggering PARP-activated systems for defense against these stresses in different eukaryotic systems remains to be tested.

Plant CBs have also been implicated in virus-host interactions, as the mechanistic properties of CBs may be hijacked by some viruses for their own benefits. For example, it has been shown that CB components, coilin and fibrillarin, can be used by viruses to mediate virus replication and spread. Detailed investigation of molecular mechanisms underlying these functions will provide new insights into our understanding of surprising multifunctional complexity of CBs.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr S.S. Makarova for communicating her unpublished results and fruitful discussions.

Funding

This work was financially supported by Scottish Government Rural and Environmental Science and Analytical Services Division (A.J.L., N.P. and M.E.T.), the State Basic Research Program of China (2014CB138403; C.Y., J.C.) and the Royal Society – Russian Foundation for Basic Research (16-54-10057-RS_a).