**5. Symplasmic conductivity in seeds**

Compartmentalization of symplasmic conductivity may differ between seeds of different plant varieties. The funiculus phloem reaches a phloem-unloading domain at the chalazal area of the outer integument, which either alone or together with the inner integument evolves to the seed coat, which differs between seed species as well. Using fluorescent probes, it has been shown that the entire outer integument at the end of the vascular bundle sheath of the funiculus is an extended symplasmic domain in *Arabidopsis thaliana* [99], and in *Crassulaceae* seeds [100], the inner integument and the embryo are separated symplasmic domains. In legumes, <sup>14</sup>C-labeled photosynthates showed PD connections between SE of the chalazal vein and the parenchyma cells [101] that comprise most of the seed coat layers [37] and consist of chlorenchyma, ground parenchyma, and branched parenchyma. In peas (*Pisum sativum* L) phloem unloading occurs in the ground parenchyma [37]. The phloem mobile molecule pyranine was transported to the chlorenchyma but not to the branched parenchyma or to the epidermis [37]. Compartmentalization of the symplasmic domains raises the important question regarding transport of viruses in seeds. This question was primarily addressed in the study of seed transmission in pea cultivars susceptible to PSbMV infection.

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

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

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

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 retain-

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].

Compartmentalization of symplasmic conductivity may differ between seeds of different plant varieties. The funiculus phloem reaches a phloem-unloading domain at the chalazal area of the outer integument, which either alone or together with the inner integument evolves to

[83]. Once in the SE, the viruses are transported by diffusion.

of a host gene in promoting *Tobamovirus* loading into SE [88, 89].

ing the C-terminus is essential for high particle accumulation [95].

**5. Symplasmic conductivity in seeds**

methyl esterase (PME) [93].

240 Advances in Seed Biology

Apparently, incidences of seed transmission of a specific virus vary between plant species and between cultivars. Comparison between cultivars allowed to delineate the sequence of events necessary for seed transmission. Infection of the vegetative tissues and of the maternal testa occurred irrespective of the capability of the virus to be transmitted via seeds. High incidence of seed transmission occurred in direct relation to virus invasion of an immature embryo.

PSbMV embryo invasion occurred at a post-fertilization stage. Pollen grains and maternal integuments were devoid of the virus. It has been suggested that the embryo, at early developmental stage, may have the symplasmic connections necessary for viral seed transmission. Immediately after fertilization zygotic divisions form a globular terminal cell and a suspensor. The integuments of the ovule develop into the testa. In the testa there are vascular strands that are continuous with the vascular bundle sheath of the funiculus. The suspensor consists of globular cell/cells that support the embryo and elongated basal cells that are in contact with the testa at the micropylar region. Using immunohistochemistry technique with antibodies raised against PSbMV particles, and in situ hybridization technique with an RNA probe specific for the viral RNA, it was shown that before fertilization PSbMV was localized in vascular tissues. Fertilization triggered invasion of the virus into the ovule along the vascular strand in both seed-transmitting and seed-un-transmitting cultivars. However, early developmental stages in seed-transmitting cultivars characteristically showed PSbMV release from vascular tissues to neighboring tissues infecting the micropyle area, concomitant to diminishing presence in the earlier infected tissues. This observation raises the question regarding the possible symplasmic connections between the maternal and filial tissues at the micropyle area. PSbMV cylindrical inclusions, which are commonly found at PD openings and were observed at the boundary between the testa and endosperm [102, 103], may only indicate the possible presence of such symplasmic connections. Crossing this boundary however does not ensure embryo invasion. It has been suggested that the suspensor is the conduit for viral transmission to the embryo [104], and pore-like structures that exist in the suspensor sheath wall at the boundary between the endosperm and the suspensor allow this transmission to occur [103].

High efficiency of PSbMV seed transmission occurred early in embryo development [105]. At this stage, the suspensor may establish continuity between virus present in the embryonic sac fluid, as was observed at late heart stage of embryo development, and the embryo [104, 105]. Viral contamination of the suspensor occurred in embryos at the globular to early heart stage [104]. The site of contact between the suspensor middle cell and the embryo became infected, further supporting the role of the suspensor in viral transmission to the embryo. The short time span the virus has before the suspensor degenerates and the fact that only passive accumulation of the virus and not its replication determines the amount of virus ready for transmission to the embryo [103, 104] are limiting conditions that hinder direct viral invasion of the embryo.

Direct viral invasion of the embryo differs from indirect embryo invasion in which the viruses infect gametes. Unlike the distribution of viruses that directly invade the embryo, such as PSbMV, viruses indirectly invading the embryo, e.g., *Pea early-browning virus* (PEBV), cause infection to the entire embryo [106, 107]. Apparently, indirect embryo invasion depends on the virus capability to invade the meristem [16]. In the plant meristem, there is a defense mechanism that protects the plant from viral RNA invasion to the germline [108]. Suppression of RNA silencing by the virus could allow meristem entry [108]. However, interfering with the plant systemic posttranscriptional gene silencing does not ensure meristematic invasion of the virus. Moreover, viral induction of DNA methylation associated with induction of posttranscriptional gene silencing does not correlate with viral invasion to meristematic cells [109]. Apparently, the signaling pathway for meristematic exclusion of viruses is not quite clear. Interestingly, the possibility that tobamoviruses could be transmitted indirectly to seeds through gamete infection was shown by mechanically applying CGMMV-infected pollen onto healthy flowering plants [110]. Under those specific experimental conditions, virus transmission rate via seeds was ~70%, which is not typical to seed transmission of tobamoviruses (**Table 1**).



**Table 1.** Selected seed-borne tobamoviruses.

**Seed**

+ + + +

+

+ +

+

*Cucurbitaceae* +

*Cucurbitaceae* +

*Solanaceae* +

*Plantaginaceae* +

*Orchidaceae*, *Solanaceae*

**Hosts Contamination % Transmission % Reference**

stage [104]. The site of contact between the suspensor middle cell and the embryo became infected, further supporting the role of the suspensor in viral transmission to the embryo. The short time span the virus has before the suspensor degenerates and the fact that only passive accumulation of the virus and not its replication determines the amount of virus ready for transmission to the embryo [103, 104] are limiting conditions that hinder direct viral invasion

Direct viral invasion of the embryo differs from indirect embryo invasion in which the viruses infect gametes. Unlike the distribution of viruses that directly invade the embryo, such as PSbMV, viruses indirectly invading the embryo, e.g., *Pea early-browning virus* (PEBV), cause infection to the entire embryo [106, 107]. Apparently, indirect embryo invasion depends on the virus capability to invade the meristem [16]. In the plant meristem, there is a defense mechanism that protects the plant from viral RNA invasion to the germline [108]. Suppression of RNA silencing by the virus could allow meristem entry [108]. However, interfering with the plant systemic posttranscriptional gene silencing does not ensure meristematic invasion of the virus. Moreover, viral induction of DNA methylation associated with induction of posttranscriptional gene silencing does not correlate with viral invasion to meristematic cells [109]. Apparently, the signaling pathway for meristematic exclusion of viruses is not quite clear. Interestingly, the possibility that tobamoviruses could be transmitted indirectly to seeds through gamete infection was shown by mechanically applying CGMMV-infected pollen onto healthy flowering plants [110]. Under those specific experimental conditions, virus transmission rate via seeds was ~70%, which is not typical to seed transmission of tobamovi-

> + + + + +

+ NT

+ NT + NT [148]

+ NT +

− +

*Solanaceae* + NT NT NT [3]

2% NT 8% <1% 0.9%

NT NT

NT NT (0.8–7.5%)

0% NT [141] [148] [144] [34] [147]

[148] [144]

[148] [3] [123]

[145] [148]

84% NT NT 95% 100%

NT NT

NT NT NT

NT NT

**Species/ acronym**

ruses (**Table 1**).

of the embryo.

242 Advances in Seed Biology

*Cucumber green mottle mosaic virus* (CGMMV)

*Kyuri green mottle mosaic virus* (KGMMV)

*Odontoglossum ringspot virus* (ORSV)

*Paprika mild mottle virus* (PaMMV)

*Pepper mild mottle virus* (PMMoV)

*Ribgrass mosaic virus* (RMV)
