through its cytoplasmic sleeve formed between the plasma membrane and the endoplasmic reticulum (ER) membrane. It is well documented that the plasmodesmal trafficking system is essential for intercellular communication medi-ated by endogenous macromolecules such as non-cell-autonomous proteins (NCAPs), RNA-silencing signals, and messenger RNAs (Dunoyer et al. 2005; Ishiwatari et al. 1998; Kurata et al. 2005; Lucas et al. 1995; Perbal et al. 1996; Wu et al. 2002), suggesting that plants have evolved mechanisms for PD-mediated cell-to-cell traffick-ing of developmental signals. In most cases, PD function as gateways to have a control over the movement signals and also their own structure. However, studies over the past few decades on plant pathogen infection have contributed for the important insights that plant viruses have evolved with mechanisms to exploit such host-derived trafficking systems when spreading one cell to another. Viruses, the obligate parasites, can only use host organisms to multi-ply their genomes. The intimate interactions between the viral genomes/genome-encoded products and host cellu-lar factors including the host transcriptional, translational and macromolecular trafficking machineries determine the success of viral infection. Studies on viral infection in plant hosts have undoubtedly exemplified the localization and identification of specific viral and host participants at PD during viral local cell-to-cell spread from the initially infected cell followed by vascular-mediated spread to dis-tant plant tissues. Such a groundbreaking discovery is that the 30 kDa movement protein (MP) encoded by Tobacco mosaic virus (TMV) was required for viral cell-to-cell movement (Deom et al. 1987; Meshi et al. 1987) and that the TMV MP was capable of modifying the size exclusion limit (SEL) of PD (Wolf et al. 1989). Subsequently, MPs and other viral proteins were reported to have a role for or be involved in the cell-to-cell and long-distance movement
Abstract Plant viruses utilize plasmodesmata (PD), unique membrane-lined cytoplasmic nanobridges in plants, to spread infection cell-to-cell and long-distance. Such invasion involves a range of regulatory mechanisms to tar-get and modify PD. Exciting discoveries in this field sug-gest that these mechanisms are executed by the interaction between plant cellular components and viral movement proteins (MPs) or other virus-encoded factors. Striking working analogies exist among endogenous non-cell-auton-omous proteins and viral MPs, in which not only do they all use PD to traffic, but also they exploit same regula-tory components to exert their functions. Thus, this review discusses on the viral strategies to move via PD and the PD-regulatory mechanisms involved in viral pathogenesis.
Plasmodesmata (PD), the fundamental unit of multicellu-larity in plants, are unique nanochannels bridging the two neighboring cells through their membrane and cytoplas-mic continuity. PD permit the passage of small molecules such as ions, hormones, photosynthates, and other nutrients
Plasmodesmata: Function and Diversity in Plant Intercellular Communication
D. Kumar, R. Kumar, T. K. Hyun equally contributed to this work.
D. Kumar · R. Kumar · T. K. Hyun · J.-Y. Kim (*) Division of Applied Life Science (BK21plus), Department of Biochemistry, Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, 27-306, 501 Jinju-Daero, Jinju 660-701, Koreae-mail: [email protected]
J Plant Res
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of many other viruses (Ding et al. 1995; Rojas et al. 1997; Schmitz and Rao 1996), indicating that plants have also evolved with a range of trafficking machineries utilized by diverse viruses. This review updates on the viral strat-egies to move via PD and the PD-regulatory mechanisms involved in viral pathogenesis.
Viral strategies for movement via plasmodesmata
A major portion of plant viruses spreads as the form of ribonucleoprotein complex (RNP) from cell to cell through PD to create a local infection, and is loaded into the phloem vasculature for long-distance transport (Benitez-Alfonso et al. 2010). The spreading potential of a given virus is an important factor determining its virulence and patho-genicity. For the successful transport of viral genomes from infected cells to neighboring cells, plant viruses encode one or more viral proteins such as MPs and coat proteins (CPs), that bind to viral RNA, and they increase the SEL of PD to facilitate the passage of the viral genome (Ding et al. 1995; Fujiwara et al. 1993; Lucas et al. 2009; Oparka et al. 1997; Roberts et al. 2001). MPs are classi-cally defined as plant virus-encoded factors that interact with PD to mediate intercellular spread of virus infection. Although all plant viruses encode MPs, there is no exten-sive sequence similarity between MPs, with only one common conserved motif (LXDX50-70G), belonging to different plant virus taxonomic group (Cheng et al. 1998; Hyun et al. 2011; Waigmann et al. 2004). In addition, their number, their interaction with each specific host factors and their mode of action are very dependent on the viral group (Scholthof 2005; Waigmann et al. 2004). The struc-turally and mechanistically diverse MPs employ at least two different movement strategies, non-tubule-guided and tubule-guided movement. The major difference between these mechanisms is whether MPs form tubules for the structural modification of PD. Tubule-guided movement was found to be common among single-stranded (ss) RNA viruses (e.g., como-, nepo-, olea-, bromo-, and trichovi-ruses), ssDNA viruses (e.g., tospoviruses), dsDNA viruses (e.g., caulimoviruses and badnaviruses) (Cheng et al. 1998; Niehl and Heinlein 2011; Perbal et al. 1993; Storms et al. 1995; van Lent et al. 1991). Tubule-guided transport involves the structural modification of PD due to the inser-tion of a tubule assembled by viral MPs that mostly results in disappearance of desmotubule in these modified PD and in dilation of PD pore (Kasteel et al. 1996; Wellink et al. 1993). However, tobamo-, diantho-, beny-, tobra-, tombus-, and hordeiviruses encoding MPs that do not form tubules use cellular machinery parallel to nuclear transport (Niehl and Heinlein 2011). A firstly identified MP was TMV MP that showed binding with ssRNA or ssDNA in a sequence-independent manner (Citovsky et al. 1990). The increasing
evidences support that TMV MPs form complexes with the transported genomes, TMV RNA, and these complexes move throughout the cell using cytoskeletal network, asso-ciate with the cell wall pectin methylesterases, and increase plasmodesmal permeability (Chen et al. 2000; Otulak and Garbaczewska 2011; Tzfira et al. 2000; Waigmann et al. 2004;). The core region in TMV MP is surrounded by two single-stranded nucleic acid binding-domains (Citovsky et al. 1992) and a domain involved in PD targeting and SEL increasing (Boyko et al. 2000; Waigmann et al. 1994). It has been claimed that the unstructured C-terminal region regulates access to those functional domains, and proved that the C-terminal region of TMV MP harbors three phos-phorylation sites (Ser258, Thr261 and Ser265), which play a role in down-regulation of MP biological activity (Cit-ovsky et al. 1993; Waigmann et al. 2000). This indicates that phosphorylation event is involved in the regulatory mechanism for controlling the interaction between TMV MP and PD in a host-dependent fashion (Waigmann et al. 2000). Likewise, the interaction of TMV-MP and PD com-ponents is explored increasingly as the number of studies has focused on finding out the endogenous plant proteins to be potential candidates to facilitate cell-to-cell spread of TMV viruses. Among them, cytoskeletal elements, cel-lular kinases and PD-localized proteins have been shown by experimental evidences to mediate TMV infection. In following sections, we will discuss on the viral strategies to move via PD in TMV, Potexvirus and Grapevine fanleaf virus (GFLV).
TMV MP and its partners in cell-to-cell movement
Ever since TMV MP, the first plant viral protein, was shown to bind with ssRNA or ssDNA (Citovsky et al. 1990), numerous studies have subsequently demon-strated that TMV MP accumulates in PD, increases their SEL (Atkins et al. 1991; Ding et al. 1992; Heinlein et al. 1998), localizes to the ER and cytoskeletal elements (Hein-lein et al. 1998; McLean et al. 1995), and is phosphoryl-ated by cellular kinases (Kawakami et al. 2003; Waigmann et al. 2000). Microtubule (MT) was considered to be a prime candidate for facilitating the transfer of the TMV viral RNA from replication sites to the PD (Heinlein et al. 1995; McLean et al. 1995; Padgett et al. 1996). When TMV MP was modified by a point mutation in its tubu-lin motif that mediates the association of MP with MT, it exhibited reduced cell-to-cell spread, and did not interact with MT (Boyko et al. 2000). Although MT may facilitate the sequestration of TMV MP for removing excessive MP from the ER (Ashby et al. 2006), this finding suggests that the interaction between TMV MP and MT is necessary for movement of the viral replication complexes to the corti-cal actin-ER network (Beachy and Heinlein 2000; Lucas
et al. 2009). However, a direct involvement of MT in TMV MP/viral RNP complex delivery to PD might be unlikely, because, a DNA-shuffled vector expressing a GFP tagged, mutagenized TMV MP (MPR3) that remained bound to the vertices of the cortical ER resulted for MPR3 to accumu-late increasingly at PD due to the limited affinity for MT (Gillespie et al. 2002). Furthermore, the disruption of MT cytoskeleton by treatment of pharmacological agents like colchicines, amiprophos-methyl and oryzalin, or by silenc-ing of the alpha-tubulin gene does not only appear to inter-fere with TMV MPR3 movement, but also on the spread of wild-type TMV MP, suggesting that cell-to-cell trafficking of TMV can occur independently of MT (Gillespie et al. 2002). Similarly, the rapid intracellular and intercellular movement of the virus replication complexes (VRCs) of TMV was blocked by inhibitors of filamentous actin and myosin, but not by inhibitors of MT (Kawakami et al. 2004). These evidences collectively support the notion that MTs are very unlikely to involve in the direct delivery of vRNP complexes to PD (Lucas 2006).
A range of experimental approaches has been employed to establish that PD function is also under the control of regulation of microfilaments. Co-injection experiments in tobacco, involving fluorescent dyes and an actin polymeri-zation inhibitor cytochalasin D, established that the disrup-tion of actin-filaments causes an increase in PD SEL (Ding et al. 1996). In addition, the plants treated with an actin polymerization inhibitor latrunculin B exhibited a reduc-tion of both MP particle trafficking and MP accumulation in PD (Sambade et al. 2008; Wright et al. 2007), indicating a role of microfilaments in viral protein and RNA complex transport. Notwithstanding the importance of actin for virus movement (Genoves et al. 2010; Hofmann et al. 2009), it has also been shown that the binding of TMV MP with G- and F-actin leads to an inhibition of actin polymeriza-tion and severing of F-actin (Su et al. 2010). Interestingly, the F-actin severing ability by TMV MP did not occur in cytoplasm, suggesting that the transport form of MPs might inhibit severing activity until they reached the PD (Su et al. 2010). Thus, it is conceivable that reorganization of the cytoskeleton by viral MPs results in a change of PD struc-ture and a concomitant increase in the SEL, providing an important insight into the cooperative role of MP, MT and microfilaments in intercellular virus movement (Fig. 1a; Aaziz et al. 2001; Boyko et al. 2000; Gillespie et al. 2002; Harries et al. 2010; Ouko et al. 2010; Sambade et al. 2008; Su et al. 2010).
An another body of evidence for the PD-mediated cell-to-cell spread of TMV MP comes from the study on the ability of TMV MP to interact with host pectin methyl esterases (PMEs) which are present in the wall around PD and in other regions of the wall in flax and tobacco (Chen et al. 2000; Morvan et al. 1998). Binding experiments
using amino acid deletion mutants of TMV MP identi-fied an MP domain required for binding to PME. Dele-tion of this region blocked the MP ability to mediate the spread of viral infection, suggesting the role of MP–PME binding in the TMV cell-to-cell movement (Chen et al. 2000). This study was extended to obtain direct sup-port that PME also plays a role in systemic spread. In the mutant plants that shows lower PME expression than the wild-type plants, the systemic spread of TMV was signif-icantly delayed (Chen et al. 2000). A range of MPs from other plant viruses, Turnip vein clearing virus (TVCV) and Cauliflower mosaic virus, were also shown to interact with PMEs. However, although it is known that (1) PMEs are associated with cell wall-embedded PD, (2) MPs colocal-ize with PD, and (3) PME-like proteins bind to MPs in cell wall extracts, the likely mechanisms allowing PMEs to facilitate viral spread remain mysterious. By co-agroinjec-tion of Nicotiana benthamiana leaves with the PME gene and the TMV:GFP vector, it was shown that PMEs could stimulate virus-induced RNA silencing (inhibition of GFP production, virus RNA degradation and stimulation of siR-NAs production), suggesting a new mechanism for PMEs to control viral cell-to-cell spread using RNA silencing (Dorokhov et al. 2006). Since the inhibiting effect of PME could be abolished by overexpressing TMV MP (Dorok-hov et al. 2006), it can also be possible that the balance between PME and MP levels might be important for viral spread. This view is supported by experiments performed with transgenic tobacco and Arabidopsis plants expressing an endogenous PME inhibitor protein (PMEI), which is tar-geted to the extracellular matrix and typically inhibit PMEs by forming a specific and stable stoichiometric 1:1 complex (Lionetti et al. 2014). The transgenic plants overexpressing PMEI exhibited compromised spread for tobamoviruses including TMV (Lionetti et al. 2014), suggesting that PMEI also interacts with PME to negatively affect viral infection, most likely by interfering with PME and TMV MP bind-ing. However, how the MP–PME interactions contribute to TMV infection and do other tobamoviruses also uses PME to infect plants are to be proved with substantial evidences.
Potexvirus cell-to-cell movement
The genus potexvirus is one of the eight genera belong-ing to the family Flexiviridae (Adams et al. 2004). Unlike tobamovirus, CP of members of genus potexvirus is required for viral cell-to-cell movement (Fedorkin et al. 2001; Verchot-Lubicz 2005; Verchot-Lubicz et al. 2007). Based on these properties, potexviruses have been clas-sified as a Type II, whereas tobamovirus is belonging to Type I (Scholthof 2005). Potexviruses have monopartite, positive-sense RNA genomes containing five overlapping open reading frames (ORFs) encoding the viral replicase
(the first ORF), triple gene block proteins [TGBp] 1–3 (the central region) and CP (the final ORF). The TGBp is evo-lutionarily conserved among viruses belonging to the gen-era Potexvirus, Pomovirus, Hordeivirus, Carlavirus, Peclu-virus, Foveavirus and Allexivirus. These viruses are often described as potex-like viruses or hordei-like viruses based on their strategies for cell-to-cell movement (Verchot-Lubicz et al. 2007). Potex-like viruses need CP for cell-to-cell movement (Scholthof 2005), and encode a TGBp1 that plays as a suppressor of RNA silencing through inter-acting with RNA-DEPENDENT RNA POLYMERASE 6 (Schwach et al. 2005; Xie and Guo 2006). On the other hand, hordei-like viruses do not require CP for trafficking (Scholthof 2005), and have an independent RNA-silenc-ing suppressor rather than TGBp (Morozov and Solovyev 2003; Verchot-Lubicz 2005; Verchot-Lubicz et al. 2007). The CP is pivotal in the virus infection process, play-ing the important role in replication, virion assembly, and viral cell-to-cell and long-distance movement (Huisman et al. 1988; Santa Cruz et al. 1998). Although the potex-viruses require their CP to potentiate cell-to-cell move-ment (Baulcombe et al. 1995; Forster et al. 1992), the CP does not have the capacity to mediate its own intercellular
transport, nor that of viral RNA, via PD (Lough et al. 1998; Santa Cruz et al. 1998). The CP in potexviruses is a three-domain protein containing highly conserved central region that interacts with viral RNA (Lico et al. 2006; Shukla and Ward 1989). The N- and C-terminal regions are quite variable, and exhibit low similarity among potexviruses. N-terminal region is located at the surface of filamentous virions, and may be crucial for intersubunit interactions and virion assembly (Verchot-Lubicz et al. 2007). In Potato X potexvirus (PVX), the N-terminal region contains a serine/threonine rich region that is both glycosylated and phos-phorylated (Atabekov et al. 2001; Baratova et al. 2004; Tozzini et al. 1994). Phosphorylation of serine residues by host kinases resulted in an enhanced viral RNA translation, suggesting that phosphorylation might promote disassem-bly of the virus (Atabekov et al. 2001). However, modifica-tions like glycosylation or phosphorylation do not appear to have relevance in cell-to-cell movement of PVX (Atabekov et al. 2001; Baratova et al. 2004; Tozzini et al. 1994). Dele-tion of some C-terminal amino acid residues of CP resulted in cell-to-cell movement deficiency, but did not affect virus particle formation (Fedorkin et al. 2000, 2001; Forster et al. 1992; Lough et al. 2000), indicating that C-terminal region of potexvirus CP might be required for cell-to-cell
Fig. 1 Models describing the various mechanisms involved in PD targeting and cell-to-cell transport of viruses. a TMV MP-mediated cell-to-cell transport of viral RNA. TMV MP binds to ER, and incor-porates in ER. The microtubule network might contribute to VRC assembly through the formation of a microtubule-proximal particle. The MP–vRNA-replication complexes diffusive process is aided by ER-associated actin network. b In triple gene block (TGB)-mediated movement of Potato X potexvirus (PVX), TGBp1-viral RNA com-plexes bind to a vesicle protein complex formed together by TGBp2 and TGBp3, which associate with the endomembrane system, includ-ing perinuclear and ER membranes, for TGBp3-mediated PD tar-geting along the ER. TGBp2–TGBp3 complex also colocalizes to small motile granules (pink circles) that move along the ER network, using the actin cytoskeleton and myosin motor proteins for motility. While TGBp2 mediates the whole complex fusion with PD, TGBp3 possesses necessary information to specify it to transport via PD. Using the endocytic pathway, TGBp2 and TGBp3 are recycled from the plasma membrane to the ER after passage of the TGBp1–vRNA complex via the pore. This process of selective trafficking is achieved through the interaction of TGBp1–vRNA complex with the putative (?) plasmodesmal trafficking machinery. c Tubule-guided cell-to-cell movement of Grapevine fanleaf virus. The GFLV MP reaches PD by diffusion or by microtubule-mediated transport upon virus replication in ER membranes, whereas PDLP is delivered to the plasma mem-brane through the secretory pathway in a myosin-dependent manner, primarily the class XI myosins XI-K and XI-2 and then it reaches to PD by diffusion through the plasma membrane. At PD, the MP interacts with the PDLP for triggering MP-based tubule formation. Then the transport of virions via PD is achieved through polar tubule assembly- and disassembly-driven treadmilling of virion-associated MP. All the models have been modified from Amari et al. (2011), Haupt et al. (2005), Hyun et al. (2011), Niehl and Heinlein (2011) and Otulak and Garbaczewska (2011) (color figure online)
movement. This view is also supported by the study that involved a mutagenic analysis of PVX CP and TGBp1, and identified the CP C-terminal 10-amino-acid region and the TGBp1 NTPase/helicase motif IV as the determinants of the physical TGBp1–CP interactions that lead to PVX-RNA encapsidation and RNA translational activation (Zay-akina et al. 2008).
TGBp1 of potexvirus has been defined as an MP based on its capacity to bind 5′ untranslated region of viral RNA in a sequence-nonspecific manner (Lough et al. 1998) and as a suppressor of RNA silencing (Schwach et al. 2005). TGBp1 was also shown to possess the capacity to increase PD SEL, traffics itself via PD, and facilitates the cell-to-cell movement of viral RNA in the presence of TGBp2, TGBp3 and CP (Hyun et al. 2011; Lough et al. 2006; Tamai and Meshi 2001; Verchot-Lubicz 2005). TGBp2 and TGBp3 of potexvirus are ER-binding proteins. Amino acid sequence analyses have suggested that TGBp2 and TGBp3 have two transmembrane and one N-terminal trans-membrane domain, respectively (Krishnamurthy et al. 2003; Mitra et al. 2003). Mutations inhibiting membrane binding of TGBp2 and TGBp3 disrupted virus movement (Krishnamurthy et al. 2003; Mitra et al. 2003), indicating that ER association of these proteins might be important for virus and protein movement. The results of microinjec-tion studies suggested that TGBp1 chaperones viral RNA to PD, whereas TGBp2 and TGBp3 act as accessory factors promoting cell-to-cell movement of TGBp1/vRNA com-plex (Lough et al. 2000; Morozov and Solovyev 2003). In addition, the biolistic bombardment studies exhibited that TGBp2 and TGBp3 are not co-translocated with the com-plex across the PD (Samuels et al. 2007), whereas potexvi-rus RNP containing TGBp1 and CP moves to adjacent cells (Hsu et al. 2004; Lough et al. 2000). These findings suggest that TGBp2 and TGBp3 serve to anchor the TGBp1/vRNA complex to the vesicle surface in vesicle trafficking along the ER-microfilament pathway (Fig. 1b; Haupt et al. 2005; Jackson et al. 2009).
Tubule-guided cell-to-cell movement of Grapevine fanleaf virus (GFLV)
GFLV belongs to the genus nepovirus of the subfam-ily Comoviridae of the new family Secoviridae. RNA1 encodes a polyprotein cleaved by RNA1-encoded chymo-trypsin-like cysteine proteinase into five products, while RNA2 determines three products; 2A homing protein, 2B MP and 2C CP (Fig. 2; Andret-Link et al. 2004). Unlike TMV MP and Potexvirus MPs, GFLV MP forms virion-filled tubules that either protrude from the membrane sur-face of infected protoplasts or are embedded within highly modified PD in infected cells (Andret-Link et al. 2004; Ritzenthaler et al. 1995a, b). The MP self-assembles into
unidirectional tubules with their base embedded in the cross-walls and their tip hanging free within the cytoplasm of adjacent cell to form a pathway for the viral particles to move cell-to-cell (Andret-Link et al. 2004). In addition, CP is also needed for GFLV movement but not replica-tion, as demonstrated by RNA2 deletion mutants and chi-meric GFLV/Arabis mosaic virus RNA2 constructs (Belin et al. 1999; Gaire et al. 1999). Tubule formation by the MP of GFLV was shown to be independent of MTs or micro-filaments, but to require a functional secretory pathway (Huang et al. 2000; Pouwels et al. 2002). Therefore, the inhibition of secretory pathway by brefeldin A resulted in the disturbing tubule formation and the redistribution of the MP to multiple foci present at the periphery of proto-plast (Huang et al. 2000; Pouwels et al. 2002). Using the stably transformed tobacco BY-2 cells expressing a fusion protein GFP:GFLV MP under the control of an inducible promoter, it has been suggested that GFP:GFLV MP is transported to specific site through Golgi-derived vesicles along two different pathways; a MT-dependent pathway in normal cells and a microfilament-dependent pathway when MTs are depolymerized (Laporte et al. 2003). Therefore, membranes associated with ER-to-Golgi transport pathway are important for the function (getting at PD and support-ing virus movement) of the MP in the genus nepovirus of the subfamily Comoviridae. Once in the immediate prox-imity of PD, the MP self-assembles into tubules, possibly with the help of cellular factors that remain to be identified (Andret-Link et al. 2004). Recently, GFLV MP was shown to interact with a PD-specific host protein, receptor-like protein (PDLP). PDLP1 was transported to PD through the secretory pathway in myosin-dependent manner (Amari et al. 2011). In the PDLP triple knock-out mutant pdlp1 pdlp2 pdlp3, the viral tubule formation and cell-to-cell spread of GFLV were reduced, suggesting that PDLPs pro-vide a PD docking platform or function as a catalyst for the assembly of MP into tubules (Fig. 1c; Amari et al. 2011; Lee and Lu 2011). However, the molecular mechanism of
Fig. 2 Diagrammatic representation of genomic RNA1 and RNA2 of Grapevine fanleaf virus (Mekuria et al. 2009). P1A putative protein-ase cofactor, Hel putative helicase, VPg viral protein genome-linked, Pro 3C-like proteinase, Pol putative RNA-dependent RNA polymer-ase, HP homing protein, MP movement protein and CP coat protein. A VPg protein linked to the 5′-non coding region
MP to lead the physical reconfiguration of the PD, together with displacement of the desmotubule, still requires to be established. Although less information is available on the transport of GFLV particles, it has been argued that the cell-to-cell movement of GFLV might involve MP-CP or MP-virion interactions during both the intracellular and intratubular transport processes (Andret-Link et al. 2004; Belin et al. 1999; Carvalho et al. 2003).
Plasmodesmatal regulation and plant defense
PD can change its status in a transient manner from hav-ing ‘closed’ to ‘open’ to ‘dilated’ state (Levy et al. 2007a). These changes regulate plant symplasmic permeability, and play an important role in plant development and defense. The molecular mechanism by which virus particles or viral RNP complexes are transported through the PD pores is not fully known. However, previous studies have attempted to partially draw the model involving direct or indirect action of viral MPs and PD components. A notion that plant cytoskeletal proteins regulate the transport of virions/MPs by a dynamic process has been demonstrated by ory-zalin treatment to MP-expressing BY-2 cells (Laporte et al. 2003). This approach resulted in tubule formation at ectopic sites, thus indicating a role for MTs in assembly site selec-tion. Thus, these results, together with the previous findings that MTs are highly distributed in cytoplasm and also tubu-lin was immunolocalized to PD nanochannels (Blackman and Overall 1998; Harries et al. 2010), suggest that MTs play important role in recruiting viruses at PD and in their cell-to-cell transport. In addition, TMV MP interacts with several cytoskeleton components, such as microtubule-associated protein MPB2C (Kragler et al. 2003), microtu-bule end-binding protein 1 (Brandner et al. 2008), and actin filaments (McLean et al. 1995). Thus, it is hypothesized that plants use a common pathway involving MPB2C to regu-late the entry of viral MPs and plant endogenous NCAPs into the NCAP pathway because KNOTTED1/SHOOT-MERISTEMLESS protein interacts with the MT-associated and viral MP binding protein, MPB2C, from Nicotiana tabacum, and its homolog in Arabidopsis, AtMPB2C, and these proteins negatively regulate KN1/TMV MP associa-tion to PD and, consequently, cell-to-cell transport (Win-ter et al. 2007). A substantial finding added more flavor to the aforementioned picture because the movement of both TMV MP and Cucurbita maxima PHLOEM PROTEIN 16 (CmPP16), appeared to be regulated by their interac-tion with a potential PD receptor NtNCAPP1 (N. tabacum NON-CELL-AUTONOMOUS PATHWAY PROTEIN1) (Lee et al. 2003). In future, one could expect some more proteins to fall under this category because many research-ers are actively involving in the identification of PD struc-tural and regulatory proteins and MP-interacting factors.
Moreover, TMV MP also interacts with a protein kinase associated with PD (Lee et al. 2005), with a DnaJ-like pro-tein (Shimizu et al. 2009), a plant ankyrin repeat-containing protein (ANK) (Ueki and Citovsky 2002) and with synap-totagmin, a calcium sensor that regulates vesicle endo- and exocytosis (Lewis and Lazarowitz 2010). Interaction with ANK and PME positively contributes to TMV intercellu-lar movement and systemic movement, respectively. The possible mechanism for controlling PD permeability is the deposition and hydrolysis of callose (β-1,3-glucan) exist-ing in the surrounding of PD. During the infection of non-tubule-guided viruses, the reducing callose level around PD is correlated with enhanced infection (Iglesias and Meins 2000; Lee and Lu 2011). The silencing of β-1,3-glucanase (BG) in Nicotiana sylvestris induces the increasing cal-lose accumulation around TMV lesions and the reduc-tion of PD SEL, and results in decreasing susceptibility to TMV (Beffa et al. 1996; Iglesias and Meins 2000). In addition, the promoted callose degradation by ectopic co-expression of BG during TMV infection increases PD SEL, and facilitates intercellular movement of infectious virus via PD (Bucher et al. 2001). The infection of TMV lack-ing CP and MP (TMVΔCPΔMP) led to the callose deposi-tion around PD in tobacco (N. tabacum) plants, whereas the reduced level of callose accumulation has been observed in TMVΔCPΔMP-infected MP transgenic plants (Levy et al. 2007b). Thus, callose is accumulated as a result of plant defense mechanism for inhibiting virus spread, while MP with viral replicase regulates the degradation of the callose for opening PD. This clearly suggests that there is a negative correlation between callose accumulation and virus spread. Tubule-forming viruses seem to use a differ-ent mechanism for PD-targeting that involves the secretory pathway. One example is that the family of PD-located proteins (PDLPs) promotes the movement of viruses that use tubule-guided movement by interacting redundantly with tubule-forming MPs within PD. Genetic disruption of this interaction leads to reduced tubule formation, delayed infection and attenuated symptoms (Amari et al. 2010). The results of this study implicate PDLPs as PD proteins with receptor-like properties involved the assembly of viral MPs into tubules to promote viral movement. The MP of GFLV interacts with PDLPs that target PD via the secretory path-way. However, the exact place at PD for such interaction to happen is yet unclear. It would be interesting to know if those PDLPs and other factors function as common targets for MPs at PD.
Based on the bioinformatic analyses, about 50 BG-related genes were identified in Arabidopsis thaliana, and were classified into several clusters based on tissue-specific expression and response to stresses and hormones (Doxey et al. 2007). During fungal infection, BGs play an active antifungal role in hydrolyzing β-1,3-glucan, a major
structural component of fungal cell walls, as members of the pathogenesis related (PR)-2 group proteins (Doxey et al. 2007). Although this indicates that BGs are involved in pathogen defense mediated by its antifungal activity, viruses can recruit specific BG(s) targeted to PD during viral infection to induce the callose hydrolysis at PD (Lee and Lu 2011). This speculation has been proved recently that the constitutive PD-associated BG, AtBG_pap, but not the stress-regulated extracellular pathogenesis-related (PR)-BGs, AtBG2 and AtBG3, is directly involved in the regulation of callose at PD and cell-to-cell viral trans-port in Arabidopsis, including the spread of viruses such as TMV and TVCV (Zavaliev et al. 2013). The level of callose accumulation is mainly controlled by balance between callose synthases and BGs (Zavaliev et al. 2011). In addition, members of PLASMODESMATA CAL-LOSE BINDING PROTEINS and REVERSIBLY GLY-COSYLATED PROTEINS in Arabidopsis were identified to be associated with PD, and also to regulate the cal-lose accumulation around PD (Sagi et al. 2005; Simpson et al. 2009). In Arabidopsis, 12 genes were identified as callose synthases, designated as GLUCAN SYNTHASE-LIKE (AtGSL1 to AtGSL12) or as CALLOSE SYNTHASE (AtCalS1 to AtCalS12) according to other nomenclature (Zavaliev et al. 2011). The expression analysis of AtGSLs showed that AtGSLs are differentially regulated by devel-opmental and physiological signals (Dong et al. 2005, 2008; Chen and Kim 2009). Five AtGSLs (AtGSL2, 5, 6, 8 and 10, or AtCalS 1, 5, 9, 10 and 12) were found to be induced by SA treatment and pathogens, but not by methyl jasmonic acid (Dong et al. 2008), suggesting that there are also SA signaling pathways involved in the induction of different AtGSL genes to regulate callose deposition during pathogen infection. Loss-of-function of AtGSL5 (pmr5 and gsl5 mutations) results in low, or lack of, cal-lose accumulation during fungal and bacterial infection (Kim et al. 2005; Nishimura et al. 2003; Wawrzynska et al. 2010; Zavaliev et al. 2011). However, the constitutive up-regulation of SA-dependent systemic acquired resistance pathway in pmr5/gsl5 plants led to the resistance against pathogens (Nishimura et al. 2003). Therefore, it has been hypothesized that SA-induced accumulation of either cal-lose or AtGSL5 triggers negative feedback mechanism for repressing SA-dependent defense response during patho-gen infection (Nishimura et al. 2003). Contrastively, PD closure by the callose accumulation enhanced SA-depend-ent defense mechanism (Lee et al. 2011). Therefore, it is possible that some other GSLs are responsible for the rapid callose accumulation at PD during pathogen infection. The further identification of possible PD-specific GSL complex and mode of its regulation will provide insight into the mechanisms of PD regulation by callose turnover during pathogen infection.
Concluding remarks
A paradigm that have already evolved from our so-far understandings on host mechanisms for both viral MPs and endogenous NCAPs is highlighted by the likely com-monalities in such regulated movement. Given the array of technical advances in genomics and proteomics, it would not be surprising to know that PD is viewed as the major means to directly or indirectly regulate plant innate immu-nity. It is especially illuminating that the ways viral MPs have adopted to spread their infection are probably through changing their size and structure of their movement forms facilitated by putative trafficking pathway components. However, currently not much is known as to how the viral MPs control host trafficking machinery, and whether host machinery, in response, have evolved mechanisms by which PD could guard plants from pathogen infection. However, it would also be equally interesting to know if viral proteins other than MPs have also the capacity to move from cell-to-cell to spread infection. Although CPs of PVX and Cucumber mosaic virus have been reported to aid intercellular transport of their respective MPs (Lough et al. 1998; Nagano et al. 2001; Santa Cruz et al. 1998), CPs themselves do not move cell-to-cell. However, given the vast list of evolutionary battle between viruses and their plant hosts, it is possible that other viral proteins might have acquired movement capacity. With the isola-tion and characterization of host proteins underlying the NCAP pathway, it is pretty sure that the focus on elucidat-ing roles for PD structural components in viral movement will answer to the above questions in the aid of technical advances in genomics and proteomics.
Acknowledgments The support for this work was provided by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2013R1A1A2007230) and by a grant from the Next-Genera-tion BioGreen 21 Program (SSAC, Grant PJ009495), Rural Develop-ment Administration, Republic of Korea. D. K and R. K were sup-ported by a scholarship from the BK21Plus Program, the Ministry of Education Korea.
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