Abstract
Plasmodesmata (PD) are the communication channels which allow the trafficking of macromolecules between neighboring cells. Such cell-to-cell movement of macromolecules is regulated during plant growth and development; however, little is known about the regulation mechanism of PD size exclusion limit (SEL). Plant viral movement proteins (MPs) enhance the invasion of viruses from cell to cell by increasing the SEL of the PD and are therefore a powerful means for the study of the plasmodesmal regulation mechanisms. In a recent study, we reported that the actin cytoskeleton is involved in the increase of the PD SEL induced by MPs. Microinjection experiments demonstrated that actin depolymerization was required for the Cucumber mosaic virus (CMV) MP-induced increase in the PD SEL. In vitro experiments showed that CMV MP severs actin filaments (F-actin). Furthermore, through the analyses of two CMV MP mutants, we demonstrated that the F-actin severing ability of CMV MP was required to increase the PD SEL. These results are similar to what has been found in Tobacco mosaic virus MP. Thus, our data suggest that actin dynamics may participate in the regulations of the PD SEL.
Key words: plasmodesmata, size exclusion limit, movement protein, actin filaments, F-actin severing
Viral MP Increases the PD SEL by Disrupting F-Actin
In order to accelerate the spreading of the virus from cell to cell, plant viral movement proteins (MPs) increase the size exclusion limit (SEL) of the plasmodesmata (PD).1 However, the mechanisms utilized by MPs to regulate the SEL of the PD are still unknown. Several models are proposed to explain the mechanisms of plasmodesmal regulation mediated by MPs.2–4 Among these models, cytoskeletal components are suggested to play important roles in this process.
Cucumber mosaic virus (CMV) MP increases the PD SEL to move from cell to cell and potentiates cell-to-cell trafficking of CMV RNA.5–7 Ding et al. reported that actin filaments (F-actin) may be involved in the regulation of plasmodesmal transport by controlling the permeability of the PD.8 In this study we sought to investigate whether the actin cytoskeleton participates in the increase of the PD SEL induced by CMV MP via microinjection experiments with various inhibitors of actin polymerization or depolymerization. The results of these experiments demonstrate that actin filaments are indeed involved in the CMV MP-induced increase in the PD SEL. Furthermore, actin depolymerization is also required for this activity.
In vitro experiments demonstrated that recombinant CMV MP binds actin filaments directly. Further study indicated that recombinant CMV MP inhibited actin polymerization and severed actin filaments in vitro (Fig. 1A). Results of the microinjection studies demonstrated that actin depolymerization is required for CMV MP-induced increase in the PD SEL. Taken together, these results indicate that CMV MP may be more likely to sever actin filaments in order to induce the increase in the SEL of the PD in vivo.
Figure 1.
A schematic representation of a model for the mechanism by which CMV MP opens the PD by disrupting actin filaments at the neck region of the PD. (A) RNP (CM V MP and its correspondent RNA) combines to ER, and then moves into PD. (B) RNP severs microfilaments in PD, therefore enhances SEL of PD.
To address this, agroinfiltration experiments were performed. No obvious changes in the cytoplasmic actin filaments were observed. There are two possibilities to explain these results. One is that CMV MP could not sever actin filaments in vivo. Alternatively, the localization of CMV MP in the PD may restrict access to actin filaments, which is required in order to sever. To test this hypothesis, we used a mutant of CMV MP, M8,9 which localizes in the cytoplasm instead of the PD sites, but retains the F-actin severing activity in vitro. Severing of the cytoplasmic actin filaments was observed after M8 was introduced. Another CMV MP mutant, M5,6,9 which does not possess F-actin severing activity in vitro, was used to further investigate the requirement of this activity in the CMV MP induced increase in the PD SEL. Microinjection experiments showed that M5 did not increase the PD SEL. These data strongly demonstrate that CMV MP could sever the actin filaments in vivo and that this severing activity is necessary for the increase in the PD SEL induced by CMV MP. Considering the localization of the CMV MP, we propose a model that, in order to traffic from cell to cell, CMV MP increases the PD SEL by severing the actin filaments at the PD site (Fig. 1B).
Similar results were obtained from the studies of Tobacco mosaic virus (TMV) MP. Actin filaments were also found to be involved in the TMV MP-induced increase in the PD SEL. Like CMV MP, actin depolymerization was required during this process; however, TMV MP could also sever F-actin in vitro. It seems that some MPs, at least CMV MP and TMV MP, increase the SEL of the PD by disrupting F-actin in cells.
Actin Cytoskeleton and Regulation of the PD SEL
The PD has received special attention in plant science research for decades. Continued progress has been made in the identification of the molecular components of the PD.10–12 Non-cell-autonomous proteins have been reported to move between cells and the trafficking of these proteins is crucial for plant growth and development. However, we still know little about the regulation mechanism of the PD SEL during this process.
Immunolabeling technology has revealed that actin and myosin are associated with the PD.13–16 In addition, actin filaments may be involved in the regulation of the PD SEL and the disruption of actin filaments leads to an increase in the permeability of plasmodesmata.8 In our study, we demonstrated that the F-actin severing ability was important for the increase in the PD SEL induced by CMV MP. Due to its localization, we propose that CMV MP increases the SEL of the PD through severing the actin filaments at the PD.
It is thought that plant viruses utilize a similar mechanism to regulate the PD SEL. Thus, it is possible that the actin cytoskeleton at the PD site may be involved in the trafficking of macromolecules through the PD in plants. Moreover, the characteristics of CMV MP are similar to some actin binding proteins (ABPs) from both plants and animals.17–20 Further study is needed to determine whether plants use ABPs to remodel the actin cytoskeleton at the PD site to regulate the SEL of the PD.
Our study provides a possible explanation to enhance the understanding of the regulation mechanism of the PD SEL. However, there are many critical issues that still need to be addressed in order to test our model. First of all, there is still lack of convincing biochemical and cellular evidence to establish the presence of actin in the PD. Second, we failed to show the specific interaction between this kind of actin cytoskeleton and CMV MP. Additionally, it is also possible that F-actin is present near the orifice of the PD and that dynamic changes in this form of actin play a role in regulating the PD SEL. Further studies to address these questions will enhance our understanding of the regulation mechanism of the PD SEL.
Acknowledgements
This work was supported by grants from the National Basic Research Program of China (2006CB100101), the 111 Project (B06003), and the National Natural Science Foundation of China (30830058 and 30721062) to M.Y.
Footnotes
Previously published online: www.landesbioscience.com/journals/psb/article/14018
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