**4. Symplasmic viral movement**

The symplast is the conduit for virus movement from cell to cell and through the phloem vasculature. They follow source to sink transportation of photoassimilates albeit with a slower rate [36]. At early stages, seeds are sinks. Nutrients enter the seed through the vascular bundle in the funiculus that ends up in the chalazal vein, which consists of xylem strand surrounded by phloem elements [37]. The seed coat (testa) is of maternal origin and mediates the release of nutrients to the embryo. It is a development of the integument/s. Symplasmic transport that can be exploited by viruses occurs from the sieve elements of the chalazal vein into the parenchyma layer [37].

#### **4.1. Cell-to-cell movement**

Viruses exploit the plasmodesmata (PD) for their movement from cell to cell. PD are membrane-bound tunnels that interconnect the cytoplasm of adjacent cells. Flattened endoplasmic reticulum membrane or desmotubule, which is surrounded by actin and myosin molecules, traverses PD in the center and is continuous with the cortical endoplasmic reticulum (ER) [38]. The actin and myosin molecules connect the desmotubule to the plasma membrane [39]. There are simple PD and branched PD; the latter are more common in mature tissues. Conversion of simple PD to branched PD presumably involves formation of bridges between simple PD. Trafficking between cells occurs via the "cytoplasmic sleeve," which is the area of the cytoplasm between the desmotubule and the plasma membrane, and via the desmotubule that bridges between the ER of adjacent cells. The desmotubule exists in both appressed and dilated states. The neck region at both ends of the PD is frequently restricted. In experiments studying the transport of small dye molecules through the cytoplasmic sleeve, it was observed that the transport is not interrupted by cytochalasin B, an inhibitor of actin polymerization [40], indicating that this intercellular movement occurs by diffusion and the PD is rate limiting [38].

Actin and myosin localized in PD may serve as scaffold for active transport of proteins. Actin filaments are also closely associated with the ER [41, 42]. Directionality of the movement of ER-localized proteins may be attributed to myosin.

The PD has size exclusion limit (SEL) [43], which changes during development and in response to environmental stress [38]. The mobility of molecules through PD is determined by Stokes radius (Rs), which is the hydrodynamic radius that is influenced by both the molecular weight of a molecule and its side group [44]. The rate of diffusion is directly correlated to the radius of a molecule [38]. SEL is measured by injecting labeled size markers such as F-dextran [45]. Transient increases in SEL were observed, and it is commonly reduced during maturation [46, 47]. SEL differs between tissues in a plant and between plant species [38]. Actin cytoskeleton may participate in regulation of SEL. Depolymerization of actin cytoskeleton by cytochalasin widened the neck region of PD and increased SEL of tobacco PD from 1 kDa to over 20 kDa [48]. Actin filaments attached to the ER participate in controlling intracellular movement of TMV viral particles [38, 49]. The integrity of the cytoskeleton is apparently necessary for viral spread. Virus movement protein (MP) is necessary for viral spread from cell to cell [50]. Cytoskeletal components were suggested to be involved in the transport of MP to the PD site and in viral cell-to-cell movement via the PD [49, 51, 52]. However, study of TMV MP and CMV MP showed that these viral proteins have F-actin depolymerization activity that causes increase in PD SEL [53]. Since actin is required for MP targeting to PD [49], it was suggested that MP-induced F-actin degradation occurs at the orifice area [53].

The MP of many viruses and of TMV is an endoplasmic reticulum membrane protein [54, 55]. Exogenously expressed MP target the PD autonomously [56–58].

Microtubules participate in cellular distribution and long-distance movement of MP and viral replication complexes [59]. However, cytoskeleton involvement in active movement of viral particles, when the PD is dilated, predicts a fast spread of the particles between cells, in the order of seconds, which does not occur [51]. The model also predicts that in the presence of MP, cytoplasmic and ER membrane proteins will show enhanced cell-to-cell movement. This apparently did not occur when movement of cytoplasmic and ER membrane-fluorescent protein probes was studied [60], which may suggest the presence of passive diffusion of the viral complexes in the desmotubule [60].

Callose, a beta 1,3-glucan [61] deposition between the plasma membrane and the cell wall in the neck region of PD, participates in determining SEL [62, 63]. Class I beta-1,3-glucanase-deficient mutants have lower SEL and show decreased susceptibility to virus infection [64]. And targeted expression of class I beta-1,3-glucanase enhances TMV infection in tobacco plants [65].

The callose accumulation is often induced in the plants under stress conditions [64]. There is a controversy regarding the effect of TMV MP on increasing SEL [66]. It has been suggested that results showing the increase in SEL by MP depend on the technique of introducing the MP [67]. Apparently under conditions of unmodified SEL by MP, TMV-derived replicase supports the activity of the viral MP on induction of callose degradation [60] and increased the conductivity via the desmotubule. The involvement of RNA replicase in cell-to-cell movement of TMV has been observed [68]. RNA helicase domain in 126 kDa replicase protein is involved in execution of cell-to-cell movement independent of MP activity.

There are several other factors that are apparently involved in MP-induced dilation of PD and may shed a light on the mechanism of SEL modification by MP. Induction of callose degradation by the viral proteins may be mediated by plant cell proteins such as TGB12K interacting protein (TIP) that interacts with both the *Potato virus X* (PVX) movement protein TGB12K and beta-1,3-glucanase [69]. TMV MP has been shown to interact with pectin methyl transferase, which is essential for dilating PD [70, 71]. The carboxy terminus of TMV MP enhances cell-tocell movement in *N. tobacco* cv. Xanthi plants [72]. Phosphorylation at the carboxy terminus is necessary for PD gating by MP [73]. MP dilation of PD may also be mediated by interacting with the *N. tabacum* noncell autonomous pathway protein 1 (NtNCAPP1) that its mutation interfered with TMV MP-induced SEL increase [74]. It has also been shown that mutation in Arabidopsis synaptotagmin, a Ca2+-binding protein that is involved in endocytic recycling, interferes with TMV MP intercellular movement [75]. TMV MP also interacts with calreticulin, which resides in PD and sequester Ca2+ [76]. This binding may indicate the involvement of local Ca2+ levels in the control of TMV MP-induced PD dilation.

The above-illustrated role of *Tobamovirus* MP in dilating PD differs from that of MP of tubuleforming viruses that modifies PD by assembling tubules that mediate cell-to-cell viral transfer [77, 78]. And the tobamoviruses differ from the filamentous viruses that engage a homologue of Hsp70 and a complex of viral components for transport through PD [79].

#### **4.2. Phloem movement**

**4.1. Cell-to-cell movement**

238 Advances in Seed Biology

rate limiting [38].

ER-localized proteins may be attributed to myosin.

that MP-induced F-actin degradation occurs at the orifice area [53].

Exogenously expressed MP target the PD autonomously [56–58].

Viruses exploit the plasmodesmata (PD) for their movement from cell to cell. PD are membrane-bound tunnels that interconnect the cytoplasm of adjacent cells. Flattened endoplasmic reticulum membrane or desmotubule, which is surrounded by actin and myosin molecules, traverses PD in the center and is continuous with the cortical endoplasmic reticulum (ER) [38]. The actin and myosin molecules connect the desmotubule to the plasma membrane [39]. There are simple PD and branched PD; the latter are more common in mature tissues. Conversion of simple PD to branched PD presumably involves formation of bridges between simple PD. Trafficking between cells occurs via the "cytoplasmic sleeve," which is the area of the cytoplasm between the desmotubule and the plasma membrane, and via the desmotubule that bridges between the ER of adjacent cells. The desmotubule exists in both appressed and dilated states. The neck region at both ends of the PD is frequently restricted. In experiments studying the transport of small dye molecules through the cytoplasmic sleeve, it was observed that the transport is not interrupted by cytochalasin B, an inhibitor of actin polymerization [40], indicating that this intercellular movement occurs by diffusion and the PD is

Actin and myosin localized in PD may serve as scaffold for active transport of proteins. Actin filaments are also closely associated with the ER [41, 42]. Directionality of the movement of

The PD has size exclusion limit (SEL) [43], which changes during development and in response to environmental stress [38]. The mobility of molecules through PD is determined by Stokes radius (Rs), which is the hydrodynamic radius that is influenced by both the molecular weight of a molecule and its side group [44]. The rate of diffusion is directly correlated to the radius of a molecule [38]. SEL is measured by injecting labeled size markers such as F-dextran [45]. Transient increases in SEL were observed, and it is commonly reduced during maturation [46, 47]. SEL differs between tissues in a plant and between plant species [38]. Actin cytoskeleton may participate in regulation of SEL. Depolymerization of actin cytoskeleton by cytochalasin widened the neck region of PD and increased SEL of tobacco PD from 1 kDa to over 20 kDa [48]. Actin filaments attached to the ER participate in controlling intracellular movement of TMV viral particles [38, 49]. The integrity of the cytoskeleton is apparently necessary for viral spread. Virus movement protein (MP) is necessary for viral spread from cell to cell [50]. Cytoskeletal components were suggested to be involved in the transport of MP to the PD site and in viral cell-to-cell movement via the PD [49, 51, 52]. However, study of TMV MP and CMV MP showed that these viral proteins have F-actin depolymerization activity that causes increase in PD SEL [53]. Since actin is required for MP targeting to PD [49], it was suggested

The MP of many viruses and of TMV is an endoplasmic reticulum membrane protein [54, 55].

Microtubules participate in cellular distribution and long-distance movement of MP and viral replication complexes [59]. However, cytoskeleton involvement in active movement of viral particles, when the PD is dilated, predicts a fast spread of the particles between cells, in the order PD that mediate cell-to-cell movement partially mediate transport of viruses into the phloem vasculature for systemic infection. PD mediate symplasmic transport between epidermal or mesophyll cells through bundle sheath, vascular parenchyma to companion cells (CC). Between CC and the enucleated sieve elements (SE), specialized PD exist named pore plasmodesmal units (PPU), which consist of multiple channels on the CC side and a single channel facing the SE [80]. PPU have higher SEL than other PD in the phloem vasculature, but it is still not enough for viral particles to cross the boundary between CC and SE, and ribonucleoproteins cross this boundary [81]. Analyzing the form of the *Tobamovirus* CGMMV that is transported through the phloem exudate showed that the virus is in the form of virus particles [82]. Apparently particle formation is essential for long-distance movement of viruses [83]. Once in the SE, the viruses are transported by diffusion.

It has been suggested that different mechanisms are employed for virus loading and unloading from the phloem since viruses enter the phloem vasculature through all classes of veins, but virus exit is limited to major veins [84–87]. Accordingly, various mechanisms in the host plants, hindering or promoting viral long-distance movement, are differentially located in the loading and unloading sites of the phloem vasculature. The cadmium ion-induced glycinerich protein is localized to the cell wall of CC and SE, and its blocking capacity of TMV and *Turnip vein clearing virus* (TVCV) long-distance movement is attributed to callose deposition. Mutation at the host VSM1 gene blocked the entry of TVCV into the phloem indicating a role of a host gene in promoting *Tobamovirus* loading into SE [88, 89].

On the other hand, at the phloem exit, protein degradation occurs and results in viral restriction to the phloem [90]. The 26S proteasome was found in pumpkin sap and Arabidopsis thaliana [91] and aminopeptidases and proteases found in pumpkin SE sap [91, 92]. Promotion of viral exit from the phloem vasculature occurs by the *Tobamovirus*-infected plant protein pectin methyl esterase (PME) [93].

The CP of many viruses, including tobamoviruses, is required for systemic movement of the viral particles in the host plants. CP ability to assemble the viral particles is important for the long-distance movement as well as the origin of assembly, indicating the importance of virion formation for transport through the phloem [94]. Analysis of CP mutants revealed that retaining the C-terminus is essential for high particle accumulation [95].

The RNA-dependent RNA polymerase activity by the 126 and/or 183 kDa proteins of TMV is necessary for the accumulation of viral particles in the phloem vasculature. Mutations occurred in the 126 kDa protein, and the 183 kDa protein, which is the read through of the amber termination of 126 ORF, prevented the accumulation of the virus in the phloem parenchyma and CCs, and systemic invasion of the virus was hindered [96]. Comparison between the systemic infection of TMV in tobacco plants and the nonsystemic infection of *Sunn-hemp mosaic virus* (SHMV) in these plants showed as well that replicase proteins are necessary for long-distance viral movement in the phloem vasculature [72, 97]. The 126/183 replicase is active also as a suppressor of silencing, which correlates with the ability of the virus to move systemically and to cause symptoms [98].
